U.S. patent number 10,570,462 [Application Number 15/167,428] was granted by the patent office on 2020-02-25 for kits and reaction mixtures for analyzing single-stranded nucleic acid sequences.
This patent grant is currently assigned to Brandeis University. The grantee listed for this patent is Brandeis University. Invention is credited to Arthur H. Reis, Jr., John E. Rice, J. Aquiles Sanchez, Lawrence J. Wangh.
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United States Patent |
10,570,462 |
Wangh , et al. |
February 25, 2020 |
Kits and reaction mixtures for analyzing single-stranded nucleic
acid sequences
Abstract
Provided herein are kits for performing for nucleic acid
sequences.
Inventors: |
Wangh; Lawrence J. (Auburndale,
MA), Rice; John E. (Quincy, MA), Sanchez; J. Aquiles
(Framingham, MA), Reis, Jr.; Arthur H. (Arlington, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Brandeis University |
Waltham |
MA |
US |
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Assignee: |
Brandeis University (Waltham,
MA)
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Family
ID: |
43900690 |
Appl.
No.: |
15/167,428 |
Filed: |
May 27, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160258001 A1 |
Sep 8, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13503324 |
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9353407 |
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PCT/US2010/053569 |
Oct 21, 2010 |
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61309265 |
Mar 1, 2010 |
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61253715 |
Oct 21, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6818 (20130101); C12Q 1/6858 (20130101); C12Q
1/6886 (20130101); C12Q 1/6827 (20130101); C12Q
1/689 (20130101); C12Q 1/6827 (20130101); C12Q
2531/101 (20130101); C12Q 2531/107 (20130101); C12Q
2537/143 (20130101); C12Q 2565/101 (20130101); C12Q
2565/1015 (20130101); C12Q 1/6858 (20130101); C12Q
2527/107 (20130101); C12Q 2531/101 (20130101); C12Q
2531/107 (20130101); C12Q 2537/143 (20130101); C12Q
2565/101 (20130101); C12Q 2565/1015 (20130101); C12Q
1/6818 (20130101); C12Q 2527/107 (20130101); C12Q
2531/101 (20130101); C12Q 2531/107 (20130101); C12Q
2537/143 (20130101); C12Q 2565/101 (20130101); C12Q
2565/1015 (20130101); C12Q 1/6827 (20130101); C12Q
2565/1015 (20130101); C12Q 2537/101 (20130101); C12Q
2527/107 (20130101); C12Q 2600/156 (20130101) |
Current International
Class: |
C07H
21/04 (20060101); C12Q 1/6818 (20180101); C12Q
1/689 (20180101); C12Q 1/6858 (20180101); C12Q
1/6886 (20180101); C12Q 1/6827 (20180101) |
Field of
Search: |
;435/6.1,6.11,6.12,91.1,91.2,91.51,183 ;436/94,501
;536/23.1,24.3,24.33,25.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-00/18965 |
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Apr 2000 |
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WO |
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WO-2001/031062 |
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May 2001 |
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WO |
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WO-2003/054233 |
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Jul 2003 |
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WO |
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WO-2006/044995 |
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Apr 2006 |
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WO |
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Other References
Allawia et al., "Thermodynamics and NMR of Internal G-T Mismatches
in DNA," Biochemistry-US, 36: 19581-10594 (1997). cited by
applicant .
De Viedma, D.G. et al., "New real-time PCR able to detect in a
single tube multiple rifampin resistance mutations and high-level
isoniazid resistance mutations in Mycobacterium tuberculosis." J
Clin Microbiol, 40(3):988-95 (2002). cited by applicant .
El-Hajj et al., "Detection of rifampin resistance in Mycobacterium
tuberculosis in a single tube with molecular beacons," J Clin
Microbiol, 39(11): 4131-7 (Nov. 2001). cited by applicant .
El-Hajj et al., "Use of sloppy molecular beacon probes for
identification of mycobacterial species," J Clin Microbiol, 47(4):
1190-8 (Apr. 2009). cited by applicant .
European Search Report from corresponding application No.
15181440.7, dated Dec. 8, 2015. cited by applicant .
Hebert et al., "Barcoding animal life: cytochrome c oxidase subunit
1 divergences among closely related species," Proc Biol Sci,
270(Supp 1): S96-9 (Aug. 7, 2003). cited by applicant .
Li et al., "A new class of homogeneous nucleic acid probes based on
specific displacement hybridization," Nucleic Acids Res, 30(2): E5,
9 pages (Jan. 15, 2002). cited by applicant .
Livak et al., "Oligonucleotides with flurescent dyes at opposite
ends provide a quenched probe system useful for detecting PCR
product and nucleic acid hybridization," PCR Methods Appl., 4(6):
357-62 (1995). cited by applicant .
Marras et al., "Multiplex detection of single-nucleotide variations
using molecular beacons," Genet Anal, 14(5-6):151-6 (Feb. 1999).
cited by applicant .
Osborne, "Single-Molecule Late-PCR Analysis of Human Mitochondrial
Genomic Sequence Variations," PLOS One, 4(5): C5636 (Jan. 1, 2009).
cited by applicant .
Piatek et al., "Molecular beacon sequence analysis for detecting
drug resistance in Mycobacterium tuberculosis," Nat Biotechnol,
16(4): 359-63 (Apr. 1998). cited by applicant .
Pierce et al., "Linear-After-The-Exponential (LATE)-PCR: primer
design criteria for high yields of specific single-stranded DNA and
improved real-time detection," Proc Natl Acad Sci USA, 102(24):
8609-14 (Jun. 14, 2005). cited by applicant .
Sanchez et al., "Linear-after-the-exponential (LATE)-PCR: an
advanced method of asymmetric PCR and its uses in quantitative
real-time analysis," Proc Natl Acad Sci USA, 101(7): 1933-8 (Feb.
17, 2004). cited by applicant .
Sanchez et al., "Two-temperature LATE-PCR endpoint genotyping," BMC
Biotechnology, Biomed Central LTD, 6(1): 44 (Dec. 4, 2006). cited
by applicant .
Santalucia J Jr., "A unified view of polymer, dumbbell, and
oligonucleotide DNA nearest-neighbor thermodynamics," Proc Natl
Acad Sci USA, 95(4): 1460-5 (Feb. 17, 1998). cited by applicant
.
Shopsin et al., "Evaluation of protein A gene polymrophic region
DNA sequencing for typing of Staphylococcus aureus strains," J Clin
Microbiol, 37(11): 3556-63 (Nov. 1999). cited by applicant .
Tyagi et al., "Molecular beacons: probes that fluoresce upon
hybridization," Nat Biotechnol, 14(3): 303-8 (Mar. 1996). cited by
applicant .
Wangh et al., "Overcoming the Crisis of TB and AIDS," Keystone
Symposia Global Health Series, Arusha, Tanzania, Oct. 20, 2009,
www.brandeis.edu/wanghlab/talks. cited by applicant.
|
Primary Examiner: Lu; Frank W
Attorney, Agent or Firm: Foley Hoag LLP Jones; Brendan T.
Gilder; Allison
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
The present application is a Divisional application of U.S.
application Ser. No. 13/503,324, issued as U.S. Pat. No. 9,353,407,
which is a national phase application under 35U.S.C. .sctn. 371of
PCT International Application No. PCT/US2010/053569, filed on Oct.
21, 2010, which claims priority to U.S. Provisional Application
61/309,265, filed Mar. 1, 2010, and to U.S. Provisional Application
61/253,715, filed Oct. 21, 2009, each of which are herein
incorporated by reference in their entireties.
Claims
We claim:
1. A kit for analyzing at least one single-stranded nucleic acid
target sequence in a sample, comprising multiple detectably
distinguishable probe sets, each of the multiple detectably
distinguishable probe sets comprising two probes: i) a probe
labeled with a non-fluorescent quencher moiety that hybridizes to a
first region of one of the at least one single-stranded nucleic
acid target sequence, and ii) a probe labeled with a fluorescent
moiety that hybridizes to a second region of said one of the at
least one single-stranded nucleic acid target sequence adjacent to
the first region of said one of the at least one single-stranded
nucleic acid target sequence, wherein after the multiple detectably
distinguishable probe sets hybridize to said one of the at least
one single-stranded nucleic acid target sequence, the multiple
detectably distinguishable probe sets are arranged adjacent to each
other on said one of the at least one single-stranded nucleic acid
target sequence, and there is no un-hybridized nucleotide between
each of the multiple detectably distinguishable probe sets; wherein
the at least one single-stranded nucleic acid target sequence is
found in an organism, wherein at least one identical probe exists
in two sets of the multiple detectably distinguishable probe sets;
wherein upon hybridization of said probe labeled with a fluorescent
moiety to said one of said at least one single-stranded nucleic
acid target sequence in said sample in the absence of the
hybridization of said probe labeled with a non-fluorescent quencher
moiety to said one of said at least one single-stranded nucleic
acid target sequence, said fluorescent moiety from said probe
labeled with a fluorescent moiety emits a fluorescent signal,
wherein, if both the probe labeled with a non-fluorescent quencher
moiety and the probe labeled with a fluorescent moiety of at least
one probe set of the multiple detectably distinguishable probe sets
hybridize to said one of said at least one single-stranded nucleic
acid target sequence, the non-fluorescent quencher moiety from the
probe labeled with a non-fluorescent quencher moiety quenches the
fluorescent signal from the fluorescent moiety from the probe
labeled with a fluorescent moiety.
2. The kit of claim 1, wherein one probe is a part of a set of the
multiple detectably distinguishable probe sets.
3. The kit of claim 2 wherein a first set and a second set of the
multiple detectably distinguishable probe sets collectively
comprise: (A) a quencher probe comprising a non-fluorescent
quencher moiety on its one end that is the probe labeled with a
non-fluorescent quencher moiety in the first set of the two of the
multiple detectably distinguishable probe sets; (B) a first
signaling probe comprising a fluorescent moiety on its first end
and a non-fluorescent quencher moiety on its second end that is the
probe labeled with a fluorescent moiety in the first set of the
multiple detectably distinguishable probe sets and the probe
labeled with a non-fluorescent quencher moiety in the second set of
the multiple detectably distinguishable probe sets; and (C) a
second signaling probe comprising a fluorescent moiety on its first
end and a non-fluorescent quencher moiety on its second end that is
the probe labeled with a fluorescent moiety in the second set of
the two of the multiple detectably distinguishable probe sets;
wherein upon hybridization of the multiple detectably
distinguishable probe sets to said one of said at least one
single-stranded nucleic acid target sequence, the non-fluorescent
quencher moiety of the quencher probe interacts with the
fluorescent moiety of the first signaling probe and the
non-fluorescent quencher moiety of the first signaling probe
interacts with the fluorescent moiety of the second signaling
probe.
4. The kit of claim 1, wherein the probe labeled with a fluorescent
moiety in each set of the multiple detectably distinguishable probe
sets is also labeled with a non-fluorescent quencher moiety.
5. The kit of claim 1, wherein a first set and a second set of the
multiple detectably distinguishable probe sets collectively
comprise: (A) a quencher probe comprising a non-fluorescent
quencher moiety on its one end that is the probe labeled with a
non-fluorescent quencher moiety in the first set of the multiple
detectably distinguishable probe sets; (B) a first signaling probe
comprising a fluorescent moiety on its first end and a
non-fluorescent quencher moiety on its second end that is the probe
labeled with a fluorescent moiety in the first set of the multiple
detectably distinguishable probe sets and the probe labeled with a
non-fluorescent quencher moiety in the second set of the multiple
detectably distinguishable probe sets; and (C) a second signaling
probe comprising a fluorescent moiety on its first end and a
non-fluorescent quencher moiety on its second end that is the probe
labeled with a fluorescent moiety in the second set of the two of
the multiple detectably distinguishable probe sets; wherein upon
hybridization of the multiple detectably distinguishable probe sets
to said one of said at least one single-stranded nucleic acid
target sequence, the non-fluorescent quencher moiety of the
quencher probe interacts with the fluorescent moiety of the first
signaling probe and the non-fluorescent quencher moiety of the
first signaling probe interacts with the fluorescent moiety of the
second signaling probe.
6. The kit of claim 1, wherein the melting temperature of the probe
labeled with a fluorescent moiety in at least one set of the
multiple detectably distinguishable probe sets is higher than the
melting temperature of its corresponding probe labeled with a
non-fluorescent quencher moiety of the at least one set of the
multiple detectably distinguishable probe sets.
7. The kit of claim 1 wherein the concentration of the probe
labeled with a fluorescent moiety of at least one set of the
multiple detectably distinguishable probe sets is lower than the
concentration of its corresponding probe labeled with a
non-fluorescent quencher moiety of the at least one set of the
multiple detectably distinguishable probe sets.
8. The kit of claim 1 wherein, when the probe labeled with a
non-fluorescent quencher moiety and the probe labeled with a
fluorescent moiety in a probe set of the multiple detectably
distinguishable probe sets hybridize to said one of said at least
one single-stranded nucleic acid target sequence, said fluorescent
moiety in the probe labeled with a fluorescent moiety and said
non-fluorescent quencher moiety in the probe labeled with a
non-fluorescent quencher moiety in the probe set of the multiple
detectably distinguishable probe sets undergo a fluorescence
resonance energy transfer (FRET).
9. The kit of claim 1 wherein said fluorescent moiety and said
non-fluorescent quencher moiety of at least one set of the multiple
detectably distinguishable probe sets are close proximity and
interact with each other upon hybridization of the at least one set
of the multiple detectably distinguishable probe sets to said one
of said at least one single-stranded nucleic acid target
sequence.
10. The kit of claim 1 further comprising primers for amplifying
said at least one single stranded nucleic acid target sequence.
11. The kit of claim 10, wherein said amplifying said at least one
single stranded nucleic acid target sequence is performed by PCR
amplification.
12. The kit of claim 10, wherein the probes in the multiple
detectably distinguishable probe sets have melting temperatures
below the annealing temperature of at least one of the primers.
13. The kit of claim 1 wherein said multiple detectably
distinguishable probe sets comprise three or more probe sets.
14. The kit of claim 11, wherein said PCR amplification is LATE-PCR
amplification.
15. The kit of claim 1, wherein the organism is selected from
bacteria, fungi, protozoa, humans and other animals, green plants,
and blue green algae.
Description
FIELD
Provided herein are fluorescence detection methods for nucleic acid
sequences and to kits for performing such methods.
BACKGROUND
Homogeneous detection of nucleic acid sequences is well known.
Detection may include a dye, for example SYBR Green, that
fluoresces in the presence of double-stranded amplification
reaction product or a fluorescently labeled oligonucleotide
hybridization probe. For hybridization probes, "homogeneous
detection" means detection that does not require separation of
bound (hybridized to target) probes from unbound probes. Among
probes suitable for homogeneous detection are linear probes labeled
on one end with a fluorophore and on the other end with a quencher
whose absorption spectrum substantially overlaps the fluorophore's
emission spectrum for FRET quenching (5' exonuclease probes
described in, for example, Livak et al. (1995) PCR Methods Appl.
4:357-362), hairpin probes labeled on one end with a fluorophore
and on the other end with a quencher (molecular beacon probes
described in, for example, Tyagi et al. (1996) Nature Biotechnology
14:303-308), double-stranded probes having a fluorophore on one
strand and a quencher on the other strand (yin-yang probes
described in, for example, Li et al. (2002) Nucl. Acids Res. 30,
No. 2 e5), linear probes having a fluorophore that absorbs emission
from a fluorescent dye and re-emits at a longer wavelength (probes
described in, for example, United States published patent
application US2002/0110450), and pairs of linear probes, one
labeled with a donor fluorophore and one labeled with an acceptor
fluorophore that hybridize near to one another on a target strand
such that their labels interact by FRET (FRET probe pairs described
in, for example, U.S. Pat. No. 6,140,054). Detection methods
include methods for detecting nucleic acid sequences in
single-stranded targets, double-stranded targets, or both.
Nucleic acid target sequences suitable for probing can in some
instances be obtained directly by isolation and purification of
nucleic acid in a sample. In other instances nucleic acid
amplification is required. Amplification methods for use with
homogeneous detection include the polymerase chain reaction (PCR),
including symmetric PCR, asymmetric PCR and LATE-PCR, any of which
can be combined with reverse transcription for amplifying RNA
sequences, NASBA, SDA, and rolling circle amplification.
Amplification-detection methods may rely on fluorescence due to
probe hybridization, or they may rely on digestion of hybridized
probes during amplification, for example, the 5' nuclease
amplification-detection method. If a sample contains or is
amplified to contain, double-stranded target, for example, the
amplification product of a symmetric PCR reaction, but
single-stranded target is desired, separation of plus and minus
strands can be accomplished by known methods, for example, by
labeling one primer with biotin and separating the
biotin-containing product strands from the other strands by capture
onto an avidin-containing surface, which is then washed.
Certain fluorescent probes useful for homogeneous detection contain
a fluorophore-labeled strand that emits a detectable signal when it
hybridizes to its target sequence in a sample. For example, a
molecular beacon probe is single-stranded and emits a detectable
fluorescent signal upon hybridization. A ResonSense.RTM. probe is
also single stranded and signals only when hybridized provided that
the sample contains a dye, generally a SYBR dye, which stimulates
hybridized probes by FRET when the dye is stimulated. Yin-yang
probes are quenched double-stranded probes that include a
fluorophore-labeled strand that emits a detectable signal it
hybridizes to its target. FRET probe pairs, on the other hand, are
probe pairs that emit a detectable fluorescent signal when both
probes of the pair hybridize to their target sequences. Some
amplification assays, notably the 5' nuclease assay, include signal
generation caused by probe cutting to generate fluorescent probe
fragments rather than simply probe hybridization.
Certain probes that generate a signal upon hybridization can be
constructed so as to be "allele-specific," that is, to hybridize
only to perfectly complementary target sequences, or to be
mismatch-tolerant, that is, to hybridize to target sequences that
either are perfectly complementary to the probe sequence or are
generally complementary but contain one or more mismatches.
Allele-specific molecular beacon probes have relatively short probe
sequences, generally single-stranded loops not more than 25
nucleotides long with hairpin stems 4-6 nucleotides long, and are
useful to detect, for example, single-nucleotide polymorphisms.
Marras et al. (1999) Genetic Analysis: Biomolecular Engineering 14:
151-156, discloses a real-time symmetric PCR assay that includes in
the reaction mixture four molecular beacons having 16-nucleotide
long probe sequences and 5-nucleotide stems, wherein each probe is
a different color, that is, includes a fluorophore that is
detectably distinguishable by its emission wavelength, and a probe
sequence differing from the others by a single nucleotide. The
sample is analyzed after each PCR cycle to detect which color
arises and thereby to identify which of four possible target
sequences perfectly complementary to one of the probes is present
in a sample. Mismatch-tolerant molecular beacon probes have longer
probe sequences, generally single-stranded loops of up to 50 or
even 60 nucleotides with hairpin stems maintained at 4-7
nucleotides. Tyagi et al. European Patent No. 1230387 discloses a
symmetric PCR amplification and homogeneous detection assay using a
set of four differently colored mismatch-tolerant molecular beacon
probes having different probe sequences 40-45 nucleotides long and
stems 5-7 nucleotides long, to hybridize competitively to, and
thereby interrogate, a 42-nucleotide long hypervariable sequence of
mycobacterial 16S rRNA genes to determine which of eight
mycobacterial species is present in a sample. The sample is
analyzed by determining a ratio of fluorophore intensities at one
or more temperatures to identify the species that is present.
El-Hajj et al (2009) J. Clin. Microbiology 47:1190-1198, discloses
a LATE-PCR amplification and homogeneous detection assay similarly
using four differently colored mismatch-tolerant molecular beacon
probes having different probe sequences 36-39 nucleotides long and
stems 5 nucleotides long to hybridize competitively to, and thereby
interrogate, a 39-nucleotide long hypervariable sequence of
mycobacterial 16S rRNA genes to determine which of twenty-seven
mycobacterial species is present in a sample. Each of the four
probes is a "consensus probe," that is, it has a single-stranded
loop complementary to multiple species but perfectly complementary
to none of them. Genomic DNA from some 27 different species were
separately amplified, the Tm of each probe was determined by
post-amplification melt analysis, and data was tabulated. To
analyze a sample containing an unknown species, the sample was
amplified and analyzed as above. The Tm's of all four probes were
compared to the tabulated results to identify the species present
in the sample.
Multiple probes, both mismatch-tolerant and allele-specific, have
been used to interrogate multiple target sequences as well as
target sequences longer than a single allele-specific probe.
Allele-specific molecular beacon probes have been utilized to
interrogate sequences longer than one probe sequence under either
of two approaches. Piatek et al. (1998) Nature Biotechnology
16:359-363, discloses performing parallel, real-time, symmetric PCR
amplification assays, each containing one of five,
fluorescein-labeled, allele-specific molecular beacons which
together span an 81-nucleotide long sequence of one strand of the
rpoB gene core region of M. tuberculosis in overlapping fashion.
Analysis was detection of probe fluorescence intensities after each
PCR cycle. Failure of any one of the probes to hybridize to
PCR-amplified target sequence ("amplicon") and emit its fluorescent
signal was taken as an indication of drug resistance. El-Hajj et
al. (2001) J. Clin. Microbiology 39:4131-4137, discloses performing
a single, multiplex, real-time, symmetric PCR assay containing five
differently colored, allele-specific molecular beacons, three
complementary to one amplicon strand and two complementary to the
other amplicon strand, which together span an 81-nucleotide long
region of the rpoB gene core region of M. tuberculosis in
overlapping fashion. Here again, probe fluorescence intensities
were obtained, and failure of any one of the probes to hybridize
and signal was taken as an indication of drug resistance. Wittwer
et al. U.S. Pat. No. 6,140,054 discloses a multiplex symmetric PCR
assay for detecting single and double base-pair mismatches in two
sequences (C282Y and H63D sites) of the human HFE gene using a
primer pair for each site, a FRET probe pair for each site, and
rapid thermal cycling. Each probe pair includes a mismatch-tolerant
fluorescein donor probe 20-30 nucleotides in length, positioned to
hybridize to target sites of possible variations, and a Cy5
acceptor probe 35-45 nucleotides long, called the "anchor" probe,
because it remains hybridized as its companion fluorescein probe
melts off the target sequence at a melting temperature dependent on
its degree of complementarity. The probe pair for one site, the
C282Y site has a lower Tm range for wild type and mutant targets
than does the probe pair for the H63D. Each probe pair has a higher
melting Tm against its mutant target than against its wild type
target As described by Witter, the melting temperature of at least
one of the probes, typically the acceptor probe, is above the
annealing temperature of both of the primers used in a symmetric
PCR amplification, and the reaction kinetics are followed in
real-time. Following amplification, a sample is analyzed by
determining the Tm's of both probe pairs from the emissions of the
acceptor (Cy5) probes. A target sequence having a single-nucleotide
mismatch to its fluorescein-labeled donor probe, that is, a
wild-type sequence, causes the donor probe to melt at a lower
temperature, thereby lowering the melting temperature by about
5.degree. C., revealing the presence of a mismatch. The genotype of
a genome is established as either homozygous or heterozygous based
on whether a signal is observed at one or two specific temperatures
whose positions are anticipated in advance. Heterozygous genomes
have equal concentrations of two possible alleles.
Analysis of nucleic acid sequences using multiple probes for long
target sequences, whether a long single target sequence or multiple
target sequences, by the foregoing methods is limited by the amount
of information that can be obtained. In FRET-probe analysis, for
every donor probe whose melting behavior is detected, there is a
corresponding acceptor probe of high Tm that serves simply as an
"anchor" and does not interrogate the target in a detectable
fashion. Methods using molecular beacons, whether allele-specific
or mismatch-tolerant, are limited by the number of fluorophore
colors that can be distinguished in a single reaction mixture
(maximally seven or eight for some detection instruments but only
four colors for other instruments), and certain molecular-beacon
methods are limited to relatively short target sequences. U.S. Pat.
No. 7,385,043 discloses an assay intended to overcome the color
limitation. It discloses a screening assay for one among as many as
fifty or even seventy possible targets by having a probe specific
to each target, specifically an allele-discriminating molecular
beacon probe, subdividing each probe into multiple parts, and
labeling each part with a different fluorophore, to create a
multi-color code identifying each probe. Assays utilizing this
approach are complicated and, thus, expensive, because the probes
must have multiple fluorophores.
Sepsis exemplifies the need to analyze long nucleic acid target
sequences. Analysis of sepsis is further complicated by the need to
differentiate among numerous bacterial species, any of which could
be the cause of infection. There is a need for single-tube
screening assays for pathogenic infections such as sepsis,
particularly assays that can be performed in laboratories other
than high-complexity CLIA laboratories, that is, point-of-care
diagnostic laboratories located at or near the site of patient
care.
SUMMARY
In some embodiments, provided herein is a homogeneous assay method
for analyzing at least one single-stranded nucleic acid target
sequence in a sample, comprising: (a) providing a sample comprising
at least one nucleic acid target sequence in single-stranded form
and for each nucleic acid target sequence at least one detectably
distinguishable set of two interacting hybridization probes, each
of which hybridizes to the at least one target, comprising: (i) a
quencher probe labeled with a non-fluorescent quencher, and (ii) a
signaling probe that upon hybridization to the at least one target
sequence in the sample in the absence of the quencher probe emits a
signal above background, wherein, if both probes are hybridized to
the at least one target sequence, the non-fluorescent quencher of
the quencher probe quenches the signal from the signaling probe;
and (b) analyzing hybridization of the signaling and quenching
probes to the at least one target sequence as a function of
temperature, the analysis including an effect on each signaling
probe due to its associated quencher probe, including but not
limited to analyzing signal increase, signal decrease, or both,
from each signaling probe.
Another aspect provided herein is the foregoing method wherein the
signaling probes include quenched fluorophores.
Another aspect provided herein is the foregoing method wherein the
melting temperature of the signaling probe in a set is higher than
the melting temperature of an associated quenching probe.
Another aspect provided herein is the foregoing method wherein
quenching when both probes are hybridized to the target sequence is
contact quenching.
Another aspect provided herein is the foregoing method wherein at
least one nucleic acid target sequence comprises at least two
target sequences, and wherein the probe set for each target
sequence includes signaling probes that are detectably different
from the signaling probes of every other probe set.
Another aspect provided herein is the foregoing method wherein
providing the sample comprising at least one target sequence in
single-stranded form comprises amplifying the nucleic acid target
sequence(s), preferably by a LATE-PCR amplification method.
Another aspect provided herein is the use of the foregoing method
in single-tube (e.g., tube, well, etc.) screening assays to
identify which nucleic acid target sequence or sequences from a
group of multiple possible target sequences is or are present in a
sample, wherein the group of multiple target sequences comprises a
variable sequence flanked by conserved, or at least relatively
conserved sequences, and a sample of target sequence in
single-stranded form is generated by an amplification method that
generates single-stranded amplicons, for example, a non-symmetric
polymerase chain reaction (PCR) method, most preferably LATE-PCR,
using only a few pairs of primers, generally not more than three
pairs, preferably not more than two pairs and more preferably only
a single pair of primers, that hybridize to the flanking sequences,
and wherein primers and at least one set of signaling and quencher
probes, preferably at least two sets, are included in the
amplification reaction mixture.
In some embodiments, probe sets (e.g. signaling and quencher
probes) are configured to hybridize to the variable sequence and to
differentiate between multiple target sequences (e.g. in a single
sample or mixture). In some embodiments, probes hybridize with
different Tm to the variable sequences of the different target
sequences. In some embodiment, one or both probes of a probe set
(e.g. signaling and/or quencher probes) comprise different degrees
of complementarity to the variable regions of the different target
sequences. In some embodiments, a signaling probe and/or quencher
probe is configured to hybridize to the variable sequence (e.g.
overlapping the actual sequence difference) of multiple target
sequences (e.g. with different Tm to the different target
sequences). In some embodiments, a signaling probe is configured to
hybridize to the variable sequence of multiple target sequences
(e.g. with different Tm to the different target sequences). In some
embodiments, a quencher probe is configured to hybridize to the
variable sequence of multiple target sequences (e.g. with different
Tm to the different target sequences).
Another aspect provided herein is a reagent kit for use in any of
the above methods comprising primers for amplifying each of the at
least one nucleic acid target sequences and at least one probe set,
and preferably including reagents for amplifying the nucleic acid
target sequence or sequences.
Probing and analysis methods provided herein apply to samples
containing single-stranded nucleic acid target sequences. Methods
of this invention include analysis of a single sequence, analysis
of two or more sequences in the same strand, analysis of sequences
in different strands, and to combinations of the foregoing. A
single-stranded nucleic acid target sequence may be a control
sequence added to a sample. A nucleic acid target sequence may be
DNA, RNA or a mixture of DNA and RNA. It may come from any source.
For example, it may occur naturally, or the target sequence may
occur in double-stranded form, in which case the single-stranded
target sequence is obtained by strand separation and purification.
If the single-stranded nucleic acid target sequence is a cDNA
sequence, it is obtained from an RNA source by reverse
transcription.
In many instances a natural source will not contain a target
sequence in sufficient copy number for probing and analysis. In
such instances the single-stranded target sequence is obtained by
amplification, generally an amplification method that includes
exponential amplification. Useful amplification methods include
isothermal amplification methods and thermal cycling amplification
methods. The amplification reaction may generate the
single-stranded nucleic acid target sequence directly, or it may
generate the target sequence in double-stranded form, in which
event the single-stranded target sequence is obtained by strand
separation and purification, as stated above. Useful amplification
methods that may be employed include, the polymerase chain reaction
(PCR), including symmetric PCR, asymmetric PCR and LATE-PCR, any of
which can be combined with reverse transcription for amplifying RNA
sequences, NASBA, SDA, TMA, and rolling circle amplification. If
the single-stranded nucleic acid target sequence is a cDNA
sequence, the amplification method will include reverse
transcription, for example, RT-PCR. In some embodiments, when
non-symmetric amplification is utilized, probe sets are included in
the amplification reaction mixture prior to amplification to avoid
contamination.
Probe sets useful in methods provided herein include a signaling
probe and an associated quencher probe. The signaling probe is a
hybridization probe that emits a detectable signal, preferably a
fluorescent signal, when it hybridizes to a single-stranded nucleic
acid target sequence in a sample, wherein the signal is quenchable
by the associated quencher probe. The quencher probe does not emit
visible light energy. Generally, a signaling probe has a covalently
bound fluorescent moiety. Signaling probes include probes labeled
with fluorophores or other fluorescent moieties, for example,
quantum dots. In some embodiments, fluorophore-labeled probes are
preferred. One type of signaling probe is a ResonSense.RTM. probe.
A ResonSense.RTM. probe is a single-stranded oligonucleotide
labeled with a fluorophore that accepts fluorescence from a DNA dye
and reemits visible light at a longer wavelength. Use of a
ResonSense.RTM. probe involves use of a double-stranded DNA dye, a
molecule that becomes fluorescent when it associates with
double-stranded DNA, which in this case is the hybrid formed when
the probe hybridizes to the single-stranded nucleic acid target
sequence. For use of a ResonSense.RTM. probe, a DNA dye, for
example, SYBR Green or SYBR Gold, is included in the sample
containing the single-stranded nucleic acid target sequence along
with the probe set or sets. Analysis includes exciting the dye and
detection emission from the ResonSense.RTM. probe or probes.
Unbound signaling probes need not be removed, because they are not
directly excited and remain single-stranded. In some embodiments,
preferred signaling probes are quenched probes; that is, probes
that emit little or no signal when in solution, even if stimulated,
but are unquenched and so emit a signal when they hybridize to a
single-stranded nucleic acid sequence in a sample being analyzed.
Yin-yang probes are quenched signaling probes. A yin-yang probe is
a double-stranded probe containing a fluorophore on one strand and
an interacting non-fluorescent quencher on the other strand, which
is a shorter strand. When a yin-yang probe is in solution at the
detection temperature, the fluorophore is quenched. The
single-stranded nucleic acid target sequence out-competes the
quencher-labeled strand for binding to the fluorophore-labeled
strand. Consequently, the fluorophore-labeled strand hybridizes to
the single-stranded nucleic acid target sequence and signals.
Especially preferred signaling probes for some embodiments provided
herein are molecular beacon probes, single-stranded hairpin-forming
oligonucleotides bearing a fluorescer, typically a fluorophore, on
one end, and a quencher, typically a non-fluorescent chromophore,
on the other end. When the probe is in solution, it assumes a
closed conformation wherein the quencher interacts with the
fluorescer, and the probe is dark. When the probe hybridizes to its
target, however, it is forced into an open conformation in which
the fluorescer is separated from the quencher, and the probe
signals. FRET probe pairs do not meet the foregoing criteria and,
thus, are not suitable for use in this invention, because their
signaling probes, the acceptor probes, do not emit a detectable
signal upon hybridization; rather, they emit a detectable signal
only when both the donor-labeled probe and the acceptor-labeled
probe.
In quenched signaling probes, quenching may be achieved by any
mechanism, typically by FRET (Fluoresence Resonance Energy
Transfer) between a fluorophore and a non-fluorescent quenching
moiety or by contact quenching. In some embodiments, preferred
signaling probes are dark or very nearly dark in solution to
minimize background fluorescence. Contact quenching more generally
achieves this objective, although FRET quenching is adequate with
some fluorophore-quencher combinations and probe constructions.
The quencher probe of a probe set is or includes a nucleic acid
strand that includes a non-fluorescent quencher. The quencher can
be, for example, a non-fluorescent chromophore such a dabcyl or a
Black Hole Quencher (Black Hole Quenchers, available from Biosearch
Technologies, are a suite of quenchers, one or another of which is
recommended by the manufacturer for use with a particular
fluorophore). In some embodiments, preferred quenching probes
include a non-fluorescent chromophore. In some embodiments,
quenchers are Black Hole Quenchers. The quencher probe of a set
hybridizes to the single-stranded nucleic acid target sequence
adjacent to or near the signaling probe such that when both are
hybridized, the quencher probe quenches, or renders dark, the
signaling probe. Quenching may be by fluorescence resonance energy
transfer (FRET or FET) or by touching ("collisional quenching" or
"contact quenching").
FIG. 1 depicts a simple embodiment that illustrates the functioning
of probe sets in analytical methods provided herein. In this
embodiment there are two probe sets, probes 2, 4 and probes 6, 8.
Probe 2 is a signaling probe, a molecular beacon probe bearing
fluorophore 3. Probe 6 is also a signaling probe, a molecular
beacon probe bearing fluorophore 7. Fluorophores 3, 7 are the same.
Probes 4, 8 are quencher probes labeled only with Black Hole
Quenchers 5 and 9, respectively. The melting temperatures (Tm's) of
the probe-target hybrids (probes hybridized to single-stranded
nucleic acid target sequence 1) are as follows: Tm probe 2>Tm
probe 4>Tm probe 6>Tm probe 8. As the temperature of the
sample is lowered from a high temperature at which no probes are
bound, probes 2, 4, 6 and 8 bind to single-stranded nucleic acid
target sequence 1 according to their hybridization characteristics.
Probe 2, a signaling probe, binds first. FIG. 1, Panel A depicts
probe 2 hybridized to sequence 1. As the temperature of the sample
continues to be lowered, quencher probe 4 binds next, adjacent to
probe 2 such that quencher 5 and fluorophore 3 are near to one
another or touching. FIG. 1, Panel B depicts probe 4 hybridized to
single-stranded nucleic acid sequence 1 adjacent to probe 2. At
this point probe 2 is dark, or at least nearly dark. If, however,
signaling probe 6 has begun to bind, it will emit fluorescence
independently of probes 2, 4. FIG. 1, Panel C depicts probe 6
hybridized to single-stranded target sequence 1 adjacent to probe
4. Finally as the temperature continues to be lowered, probe 8 will
bind, and its quencher 9 will quench fluorescence emission from
fluorophore 7 of probe 6. FIG. 1, Panel D depicts probe 8
hybridized adjacent to probe 6. Analysis by hybridization is shown
in FIG. 1, Panel E, which depicts the increase and decrease of
fluorescence from fluorophores 3, 7 as a function of temperature.
Such curves can be obtained as annealing (hybridization) curves as
the temperature is lowered, or can be obtained as melting curves as
the temperature is increased. As the sample temperature is lowered
from 70.degree. C., fluorescence curve 10 in Panel E first rises as
probe 2 hybridizes to single-stranded nucleic acid sequence 1, then
decreases as probe 4 binds, then increases again as probe 6
hybridizes, and finally decreases to a very low level as probe 8
hybridizes. One can deduce from curve 10 that each signaling probe
has a higher Tm than its associated quencher probe.
Signaling and quenching probes useful in methods provided herein
may be allele-specific (hybridize only to a perfectly complementary
single-stranded nucleic acid target sequence in the method) or
mismatch tolerant (hybridize to single-stranded nucleic acid target
sequences containing one or more mismatched nucleotides, or
deletions or additions). In some embodiments, one probe of a set
may be allele-specific; and the other probe, mismatch tolerant.
Experiments conducted during development of embodiments provided
herein demonstrated that secondary structure of a target strand
outside the sequences to which probes hybridize can affect the
results of annealing or melting analysis. Accordingly, in some
embodiments, not every nucleotide in a nucleic acid target sequence
needs to be hybridized to a probe. For example, if the target
sequence contains a hairpin, the corresponding probe can be
designed in some cases to hybridize across the base of the hairpin,
excluding the hairpin sequence. A probe set may include an
allele-specific signaling probe and an allele-specific quencher
probe, a mismatch-tolerant signaling probe and a mismatch-tolerant
quencher probe, an allele-specific signaling probe and a
mismatch-tolerant quencher probe, or a mismatch-tolerant signaling
probe and an allele-specific quencher probe. A mismatch-tolerant
probe may be perfectly complementary to one variant of a variable
target sequence, or it may be a consensus probe that is not
perfectly complementary to any variant. Multiple probe sets may
include combinations of sets of any of the foregoing types.
Additionally, analytical methods provided herein may utilize one or
more signaling/quenching probe sets in combination with one or more
conventional probes that signal upon hybridization to their target,
for example, molecular beacon probes.
Probes useful in the methods provided herein may be DNA, RNA, or a
combination of DNA and RNA. They may include non-natural
nucleotides, for example, PNA, LNA, or 2' o-methyl ribonucleotides.
They may include non-natural internucleotide linkages, for example,
phosphorothioate linkages. The length of a particular probe depends
upon its desired melting temperature (Tm), whether it is to be
allele-specific or mismatch tolerant, and its composition, for
example, the GC content of a DNA probe. Generally speaking,
allele-specific probes are shorter than mismatch-tolerant probes.
For example, an allele-specific DNA molecular beacon probe may have
a target-hybridizing sequence, the loop, in the range of 10-25
nucleotides long, with a double-stranded stem 4-6 nucleotides long.
Mismatch-tolerant DNA molecular beacon probe may have a somewhat
longer loop, generally not more than 50 nucleotides in length, and
a shorter double-stranded stem, preferably either one or two
nucleotides long.
In some embodiments, each signaling probe has a separate quenching
probe associated with it. In some embodiments, however one probe
may be a part of two probe sets. For example, a quencher probe may
be labeled with a quencher at each end, whereby the ends interact
with different signaling probes, in which case three probes
comprise two probe sets. Also, some embodiments may utilize both
ends of a quenched signaling probe, for example, a molecular beacon
signaling probe having a fluorophore on one end and a quencher on
the other end. The fluorophore interacts with a quencher probe,
comprising one set, and the quencher interacts with a signaling
probe, comprising another set.
For analysis of a sample, the probe sets that are used are
detectably distinguishable, for example by emission wavelength
(color) or melting temperature (Tm). Making a probe set
distinguishable by Tm from other probe sets can be accomplished in
any suitable way. For example, all signaling probes in an assay may
have different Tm's. Alternatively, all signaling probes could have
the same Tm but the quencher probes could have different Tm's. Some
fluorescence detectors can resolve up to eight differently colored
fluorophores; others, only four. The same fluorescence emitter, for
example, the same fluorophore, can be used on more than one
signaling probe for a sample, if the signaling probe's can be
differentiated for detection by their melting temperatures. In
assays provided herein, Tm's should be separated by at least
2.degree. C., preferably by at least 5.degree. C. and, in certain
embodiments by at least 10.degree. C. Available temperature space
constrains the use of multiple signaling probes having the same
fluorophore. If an assay is designed for annealing and/or melt
analysis over a range of 80.degree. C. to 20.degree. C., for
example, one can utilize more probe sets sharing a color than one
can use in an assay designed for such analysis over a range of
70.degree. C. to 40.degree. C., for which one may be able to use
only 3-5 probe sets sharing a color. Using four colors and only two
probe sets sharing each color, a four-color detector becomes
equivalent to an eight-color detector used with eight probes
distinguishable by color only. Use of three probe sets sharing each
of four colors, twelve different probes sets become
distinguishable.
It is generally preferred that quencher probes have lower Tm's than
their associated signaling probes. With that relationship, the
signaling probe emits a temperature-dependent signal through the
annealing temperature range of both probes of the set as the
temperature of the solution is lowered for an annealing curve
analysis, and through the melting temperature range of both probes
of the set as the temperature of the solution is raised for a
melting curve analysis. If, on the other hand, the quencher probe
of a probe set has a higher Tm than its associated signaling probe,
the signaling probe's emission is quenched through the annealing
temperature range and melting temperature range of both probes of
the set, and no fluorescent signal is emitted for detection. This
can be ascertained by examination of the annealing curve or the
melting curve. The lack of signal provides less information about
the single-stranded nucleic acid target sequence than does a curve
of the probe's fluorescence as a function of temperature. In some
embodiments, when mismatch-tolerant probes are used for analysis of
a variable sequence, quencher probes with lower Tm's than their
associated signaling probes are used with respect to all or all but
one of the target sequence variants. If a quencher probe has a
higher Tm against only one variant, signal failure will reveal that
variant, as long as failure of the sample to include the
single-stranded nucleic acid target sequence (particularly failure
of an amplification reaction) is otherwise accounted for by a
control or by another probe set for the single-stranded nucleic
acid target sequence. Similarly, if not all variants are known,
such signal failure will reveal the presence of an unknown variant.
In some embodiments, it is preferred that in an assay utilizing
multiple probe sets for at least one nucleic acid target sequence,
the quencher probe of at least one probe set has a lower Tm than
its associated signaling probe.
Melting temperature, Tm, means the temperature at which a nucleic
acid hybrid, for example, a probe-target hybrid or primer-target
hybrid, is 50% double-stranded and 50% single-stranded. For a
particular assay the relevant Tm's may be measured. Tm's may also
be calculated utilizing known techniques. In some embodiments,
preferred techniques are based on the "nearest neighbor" method
(Santa Lucia, J. (1998), PNAS (USA) 95: 1460-1465; and Allawi, H.
T. and Santa Lucia, J. (1997), Biochem. 36: 10581-10594). Computer
programs utilizing the "nearest neighbor" formula are available for
use in calculating probe and primer Tm's against perfectly
complementary target sequences and against mismatched target
sequences. For examples in this specification, the program Visual
OMP (DNA Software, Ann Arbor, Mich., USA) was used, which uses the
nearest neighbor method, for calculation of Tm's. In this
application the Tm of a primer or probe is sometimes given with
respect to an identified sequence to which it hybridizes. However,
if such a sequence is not given, for mismatch-tolerant probes that
are perfectly complementary to one variant of a single-stranded
nucleic acid target sequence, the Tm is the Tm against the
perfectly complementary variant. In many embodiments there will be
a target sequence that is perfectly complementary to the probe.
However, methods may utilize one or more mismatch-tolerant primer
or probes that are "consensus primers" or "consensus probes." A
consensus primer or probe is a primer or probe that is not
complementary to any variant target sequence or, if not all
possible target sequences are known, to any expected or known
sequence. A consensus primer is useful to prime multiple variants
of a target sequence at a chosen amplification annealing
temperature. A consensus probe is useful to shrink the temperature
space needed for analysis of multiple variants. For a consensus
primer or probe, if no corresponding target sequence is given, the
Tm refers to the highest Tm against known variants, which allows
for the possibility that an unknown variant may be more
complementary to the primer or probe and, thus, have higher
primer-target Tm or probe-target Tm.
Assays provided herein may utilize probe concentrations that are
greater than or less than target nucleic acid concentration. The
probe concentrations are known on the basis of information provided
by the probe manufacturer. In the case of target sequences that are
not amplified, target concentrations are known on the basis of
direct or indirect counting of the number of cells, nuclei,
chromosomes, or molecules are known to be present in the sample, as
well as by knowing the expected number of targets sequences usually
present per cell, nucleus, chromosome, or molecule. In the case of
target sequences that are amplified, there are a number of ways to
establish how many copies of a target sequence have been generated
over the course of an amplification reaction. For example, in the
case of a LATE-PCR amplification reaction the number of
single-stranded amplicons can be calculated as follows: using a
signaling probe without a quencher (in the case of quenched
signaling probe that means the probe minus the quencher) in a
limiting concentration such as 50 nM and its corresponding quencher
probe in excess amount such as 150 nM, the number of cycles it
takes to decrease the fluorescence to zero (or, in practical terms,
to its minimal background level) is proportional to the rate of
amplification of single-stranded amplicons. When fluorescence
reaches zero (minimal background level), all of the signaling
probes have found their target, and the concentration of the
amplicons exceeds that of the signaling probe. Another method for
estimating amplicon concentration in a LATE-PCR amplification is
presented in Example 10 of published patent application EP 1805199
A2. In certain embodiments an amplification reaction may be
continued until the amplicon being produced reaches a "terminal
concentration." Experiments conducted during development of
embodiments provided herein demonstrated that a LATE-PCR
amplification begun with differing amounts of target tends to
produce eventually the same maximum concentration of amplicon (the
"terminal concentration"), even though amplification begun with a
high starting amount of target reaches that maximum in fewer cycles
than does the amplification begun with a low starting amount of
target. To achieve the terminal concentration beginning with a low
amount of target may require extending the amplification through 70
or even 80 cycles.
Some embodiments utilize probe sets in which the concentration of
the signaling probe is lower than the concentration of its
associated quencher probe. This ensures that, when both probes are
hybridized to their at least one nuclei acid target sequence, the
signaling probe is quenched to the greatest possible degree,
thereby minimizing background fluorescence. It will be appreciated
that background fluorescence in an assay is the cumulated
background of each signaling probe of a given color and that probes
of a different color may contribute further to background
signal.
Methods provided herein include analyzing the hybridization of
probe sets to the single-stranded nucleic acid target sequences. In
methods provided herein, hybridization of signaling probes and
quencher probes as a function of temperature is analyzed for the
purpose of identifying, characterizing or otherwise analyzing at
least one nucleic acid target sequence in a sample. In some
embodiments analysis includes obtaining a curve or, if multiple
colors are used, curves of signals from signaling probes as the
temperature of a sample is lowered (see FIG. 1, Panel E) or
obtaining a curve or curves of signals as the sample temperature is
raised, or both. It is known that the shapes of the two types of
curves are not necessarily identical due to secondary structures.
Either or both of those curves can be compared to a previously
established curve for a known single-stranded nucleic acid target
sequence as part of the analysis, for example, identifying the
single-stranded nucleic acid target sequence being probed.
Derivative curves can also be utilized to obtain, for example, the
Tm of a signaling probe against a nucleic acid target sequence. It
is not always necessary, and it may not be desirable, to utilize
entire fluorescence curves or their derivatives. In certain
embodiments analysis of the hybridization of signaling probes and
quencher probes includes obtaining fluorescence readings at one or
several temperatures as the sample temperature is lowered or
raised, where those readings reflect an effect on each signaling
probe due to its associated quencher probe. For example, if it is
desired to distinguish among known variants of a target sequence,
and one learns from hybridization curves of variants that
fluorescence at two temperatures distinguish the variants, one need
acquire fluorescence at only those two temperatures for either
direct comparison or for calculation of ratios that can be
compared. In most embodiments the analysis will include signal
increase, signal decrease, or both, from each signaling probe.
In analytical methods provided herein, provision of an at least one
nucleic acid target sequence may include nucleic acid
amplification. Some preferred methods are those which generate the
target sequence or sequences in single-stranded form. LATE-PCR
amplification of DNA sequences or RNA sequences (RT-LATE-PCR) is
especially preferred in some embodiments. LATE-PCR amplifications
and amplification assays are described in, for example, European
patent EP 1,468,114 and corresponding U.S. Pat. No. 7,198,897;
published European patent application EP 1805199 A2; Sanchez et al.
(2004) Proc. Nat. Acad. Sci. (USA) 101: 1933-1938; and Pierce et
al. (2005) Proc. Natl. Acad. Sci. (USA) 102: 8609-8614. All of
these references are hereby incorporated by reference in their
entireties. LATE-PCR is a non-symmetric DNA amplification method
employing the polymerase chain reaction (PCR) process utilizing one
oligonucleotide primer (the "Excess Primer") in at least five-fold
excess with respect to the other primer (the "Limiting Primer"),
which itself is utilized at low concentration, up to 200 nM, so as
to be exhausted in roughly sufficient PCR cycles to produce
fluorescently detectable double-stranded amplicon. After the
Limiting Primer is exhausted, amplification continues for a desired
number of cycles to produce single-stranded product using only the
Excess Primer, referred to herein as the Excess Primer strand.
LATE-PCR takes into account the concentration-adjusted melting
temperature of the Limiting Primer at the start of amplification,
Tm.sub.[0].sup.L, the concentration-adjusted melting temperature of
the Excess Primer at the start of amplification, Tm.sub.[0].sup.X,
and the melting temperature of the single-stranded amplification
product ("amplicon"), Tm.sub.A. For LATE-PCR primers, Tm.sub.[0]
can be determined empirically, as is necessary when non-natural
nucleotides are used, or calculated according to the "nearest
neighbor" method (Santa Lucia, J. (1998), PNAS (USA) 95: 1460-1465;
and Allawi, H. T. and Santa Lucia, J. (1997), Biochem. 36:
10581-10594) using a salt concentration adjustment, which in our
amplifications is generally 0.07 M monovalent cation concentration.
For LATE-PCR the melting temperature of the amplicon is calculated
utilizing the formula: Tm=81.5+0.41 (% G+% C)-500/L+16.6 log
[M]/(1+0.7 [M]), where L is the length in nucleotides and [M] is
the molar concentration of monovalent cations. Melting temperatures
of linear, or random-coil, probes can be calculated as for primers.
Melting temperatures of structured probes, for example molecular
beacon probes, can be determined empirically or can be approximated
as the Tm of the portion (the loop or the loop plus a portion of
the stem) that hybridizes to the amplicon. In a LATE-PCR
amplification reaction Tm.sub.[0].sup.L is preferably not more than
5.degree. C. below Tm.sub.[0].sup.X, more preferably at least as
high and even more preferably 3-10.degree. C. higher, and Tm.sub.A
is preferably not more than 25.degree. C. higher than
Tm.sub.[0].sup.X, and for some preferred embodiments preferably not
more than about 18.degree. C. higher.
LATE-PCR is a non-symmetric PCR amplification that, among other
advantages, provides a large "temperature space" in which actions
may be taken. See WO 03/054233 and Sanchez et al. (2004), cited
above. Certain embodiments of LATE-PCR amplifications include the
use of hybridization probes, in this case sets of signaling and
quencher probes, whose Tm's are below, more preferably at least
5.degree. C. below, the mean primer annealing temperature during
exponential amplification after the first few cycles. Sets of
signaling and quencher probes are included in LATE-PCR
amplification mixtures prior to the start of amplification. A DNA
dye, if used, can also be incorporated into the reaction mixture
prior to the start of amplification.
Amplification and detection methods provided herein enable
single-tube, homogeneous assays to detect variants of a particular
variable sequence, for example, a ribosomal RNA sequence, whose
variants are found in a group of organisms, including but not
limited to bacteria, fungi, protozoa, humans and other animals,
green plants, and blue green algae, where the particular variable
sequence is flanked by sequences that are conserved, or relatively
conserved, among members of the group. Variants of the variable
sequence can then be amplified by a primer-dependent amplification
method, preferably an amplification method that generates
single-stranded nucleic acid target sequences, such as a
non-symmetric polymerase chain reaction (PCR) DNA amplification
method, most preferably LATE-PCR (with reverse transcription, if
the variants are RNA), using only a few pairs, sometimes only a
single pair, of primers that hybridize to the flanking sequences.
Sets of signaling probes and quencher probes are included in the
amplification reaction mixture, and the amplification product or
products are analyzed by the analytical methods provided
herein.
In some embodiments, provided herein are kits comprising
combinations of signaling and quencher probes, which may be
referred to as "oligonucleotide sets," for use in the foregoing
methods, as well as kits that additionally include some or all of
primers, amplification reagents, such as amplification buffer, DNA
polymerase and, where appropriate, reverse transcriptase. Kits may
also include control reagents (e.g., positive and negative
controls) or any other components that are useful, necessary, or
sufficient for practicing any of the methods described herein, as
well as instructions, analysis software (e.g., that facilitates
data collection, analysis, display, and reporting), computing
devices, instruments, or other systems or components.
Provided herein are amplification reaction mixtures for performing
amplification assay methods of this invention. Such reaction
mixtures include reagents for providing single-stranded nucleic
acid target sequence or sequences to be analyzed, and sets of
signaling and quencher probes for the analysis. Some reaction
mixtures include reagents for non-symmetric amplification, most
preferably LATE-PCR and RT-LATE-PCR amplification.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, Panels A-D are schematics showing hybridization of two sets
of signaling and quencher probes to a single-stranded nucleic acid
target sequence in a sample as a function of temperature; and FIG.
1, Panel E, shows the fluorescence versus temperature of the
sample.
FIG. 2 is a schematic representation of a single-stranded nucleic
acid sequence from Example 1 showing probe binding locations and
primer binding locations.
FIGS. 3A and 3B present melt-curve analyses from amplifications
described in Example 1 for several strains.
FIGS. 4A-4D presents derivative melting curves for mixtures of TB
strains in various proportions as described in Example 2.
FIG. 5 is a schematic representation of a single-stranded nucleic
acid sequence from Example 3 showing probe binding locations and
primer binding locations.
FIG. 6 is a schematic representation of another single-stranded
nucleic acid sequence from Example 3 showing probe binding
locations and primer binding locations.
FIGS. 7A-7C are graphs of fluorescence versus temperature for each
of the fluorophores in the sample of Example 3.
FIG. 8 is a schematic representation of a single-stranded nucleic
acid sequence from Example 4 showing probe binding locations and
primer binding locations.
FIGS. 9A-9C are graphs of fluorescence versus temperature for each
of the fluorophores in Example 4 against one single-stranded
nucleic acid target sequence.
FIGS. 10A-10C are graphs of fluorescence versus temperature for
each of the fluorophores in Example 4 against another
single-stranded nucleic acid target sequence.
FIGS. 11A-11C are bar codes made from the fluorescence-curve data
shown in FIG. 9.
FIG. 12 is a schematic representation of a selected region of the
16s gene of several species of bacteria showing binding locations
of the primer pair and of four sets of signaling and quencher
probes used in Example 5.
FIG. 13 is a graph presenting annealing curves (fluorescence versus
temperature) of the probe sets of Example 5 following amplification
of the selected region starting with genomic DNA of different
species of bacteria.
FIG. 14 is a graph presenting the first derivative (-dF/dT) curves
of the annealing curves of FIG. 13.
FIG. 15 is a graph presenting annealing curves of the probe sets of
Example 5 following amplification of mixtures of genomic DNA
described in Example 6.
FIG. 16 is a schematic representation of a selected region of the
16s gene of Acinetobacter baumanii showing binding locations of the
primer pair and four sets of signaling and quencher probes used in
Example 7.
FIG. 17A is a graph presenting annealing curves of the Cal Red 610
fluorophore following amplification of different target species in
Example 7.
FIG. 17B is a graph presenting annealing curves of the Quasar 670
fluorophore following amplification of different target species in
Example 7.
FIG. 18 is a graph presenting the annealing curves (fluorescence
versus temperature) for the amplification reaction described in
Example 4.
FIG. 19 is an illustrative graph showing how an annealing curve
(Fluorescence versus temperature) of a reaction such as described
in Example 4 shifts with increasing concentration of target
molecules.
FIG. 20 is a graph presenting normalized melting curves
(fluorescence temperature) of the probe set of Example 9 following
amplification of homozygous SNP alleles and heterozygous
mixture.
FIG. 21 is a graph presenting the first derivative (-dF/dT) curves
of the melting curves of FIG. 20.
FIG. 22 is a graph presenting the first derivative (-dF/dT) of
post-amplification annealing curves of twelve MRSA samples using
the single set of ON/OFF probes described in Example 10.
DETAILED DESCRIPTION
In some embodiments, useful signaling probes are hybridization
probes that emit a detectable signal above background when they
hybridize to a target sequence. Some preferred signaling probes are
quenched probes, that is, probes whose fluorescence is quenched
when the probes are in solution. In some embodiments, signaling
probes are molecular beacon probes, which are single-stranded
oligonucleotides that have a covalently bound signaling fluorophore
one end and a quencher moiety, for example another fluorophore,
preferably a non-fluorescent quencher, for example Dabcyl or a
Black Hole Quencher, on the other end. Molecular beacon probes have
a central target-complementary sequence flanked by arm sequences
that hybridize to one another in the absence of the target
sequence, causing the probe to adopt a stem-loop conformation in
which the quenching moiety quenches fluorescence from the signaling
fluorophore by fluorescence resonance energy transfer (FRET) or by
collisional (or contact) quenching. Molecular beacon probes have
low background fluorescence due to efficient quenching in the
stem-loop structure. When the target-complementary sequence, that
is, the loop or the loop plus some or all of the stem nucleotides,
hybridizes to a target sequence, the arm sequences are separated
from one another, and the probe's quenching moiety no longer
quenches fluorescence from the signaling fluorophore. See Tyagi and
Kramer (1996) Nature Biotechnology 14: 303-308; and El-Hajj et al.
(2001) J. Clin. Microbiology 39: 4131-4137. Other types of
oligonucleotide hybridization probes that emit a detectable
fluorescent signal upon hybridization may also be used. Such
include, for example, single-stranded linear probes labeled at
opposite ends with a signaling fluorophore and a quencher
fluorophore. (see, Livak et al. (1995) PCR Methods Appl. 357-362);
double-stranded oligonucleotide probes having a signaling
fluorophore on one strand and a quenching moiety on the other
strand (see Li et al. (2002) Nucl. Acid. Res. 30(2)e5); and
ResonSense.RTM. probes, linear single-stranded probes labeled with
a signaling fluorophore that emits energy received by FRET from a
DNA dye such as SYBR Green that associates with the probe-target
hybrid (see U.S patent publication US 2002/0119450).
Quencher probes may be structurally similar to signaling probes but
without a signaling fluorophore, that is, with just a quencher
moiety. Because quencher probes do not contribute background
fluorescence, they can be linear probes. For a quencher probe to be
"associated" with a signaling probe, that is, to be able to quench
that signaling probe when both are hybridized to the
single-stranded nucleic acid target sequence being analyzed, the
signaling fluorophore of the signaling probe is located at or near
the end nearest the quencher probe, and the quenching moiety of the
quencher probe is located at or near the end of the quencher probe,
such that that fluorophore and that quenching moiety can interact
by FRET or by contact quenching. In some embodiments, quenching
moieties for quencher probes are non-fluorescent chromophores such
as Dabcyl and Black Hole Quenchers.
Signaling probes and quenching probes may be either
sequence-specific or mismatch tolerant. A sequence-specific probe
hybridizes in the assay only to a target sequence that is perfectly
complementary to the probe. A mismatch-tolerant probe hybridizes in
the assay, not only to a target sequence that is perfectly
complementary to the probe, but also to variations of the target
sequence that contain one or more mismatches due to substitutions,
additions or deletions. For mismatch-tolerant probes, the greater
the variation of the target from perfect complementarity, the lower
the Tm of the probe-target hybrid. Combinations of
sequence-specific and mismatch-tolerant probes may be used in a
single assay. If a probe is sequence-specific, any mismatch in the
target sequence will cause the probe not to hybridize, and its lack
of hybridization will show in the melt curve and the derivative
curve. For example, if a signaling probe hybridizes, causing an
increase in fluorescence, but its associated quencher probe does
not hybridize, fluorescence will not decrease as the temperature is
lowered through the Tm of the quencher probe, revealing that the
quencher probe did not hybridize and indicating a target mutation
in the sequence complementary to the quencher probe. That is a
satisfactory result, if one wishes to determine whether or not
there is any mutation. That is not satisfactory, however, if one
wishes to determine which one of several possible mutations of that
sequence is present. For that, it is preferable that the associated
quencher probe be mismatch tolerant, so that different mutations
can be distinguished by their different effects on the melting
curve (and derivative curve) due to differing Tm effects of
different mutations.
In some preferred embodiments, a signaling probe of a set has a
higher Tm with respect to the single-stranded nucleic acid target
sequence than does its associated quencher probe. With that
relationship, as a sample is subjected to melt analysis, for
example, as temperature is increased signal first increases as the
quencher probe melts off and then decreases as the signaling probe
melts off. With the opposite relationship, signal remains quenched
as the lower Tm signaling probe melts off and does not then
increase as the higher Tm quencher probe melts off. The preferred
relationship thus provides more information. In some embodiments,
it is preferred that the quencher probe of a set reduces the signal
from its associated signaling probe to a very large extent. In such
embodiments, it is preferred that the concentration of the quencher
probe equal or exceed the concentration of the signaling probe. In
order to maximize signal amplitude, certain embodiments utilize
probe concentrations that are in excess with respect to the
single-stranded nucleic acid target sequence, thereby ensuring that
all or nearly all copies of the target sequence will have
hybridized probes.
Methods provided herein include the use of a single set of
interacting signaling and quencher probes. Methods also include the
use multiple sets of interacting signaling and quencher probes,
wherein each signaling probe is detectably distinguishable from the
others. Distinction of fluorescent probes may be by color (emission
wavelength), by Tm, or by a combination of color and Tm. Multiple
sets of interacting probes may be used to interrogate a single
target sequence or multiple target sequences in a sample, including
multiple target sequences on the same target strand or multiple
target sequences on different strands. Multiplex detection of
multiple target sequences may utilize, for example, one or more
sets of signaling/quencher probes specific to each target sequence.
In some embodiments, multiplex methods utilize a different
fluorescent color for each target sequence. Certain embodiments
utilize the same color for two different target sequences,
available temperature space permitting.
In some embodiments, methods comprise analyzing hybridization of
signaling/quencher probe sets to one or more single-stranded
nucleic acid target sequences as a function of temperature. Signal,
preferably fluorescent signal, from the signaling probe or probes
may be acquired as the temperature of a sample is decreased
(annealing) or increased (melting). Analysis may include
acquisition of a complete annealing or melting curve, including
both increasing and decreasing signals from each signaling probe,
as is illustrated in FIG. 1, Panel E. Alternatively, analysis can
be based only on signal increase or signal decrease. Analysis may
utilize only signals at select temperatures rather than at all
temperatures pertinent to annealing or melting. Analysis at some or
all temperatures may be digitized to create a signature for a
target sequence, for example, a bar code such as described in
Example 4 and shown in FIG. 11. Analysis may include comparison of
the hybridization of an unknown single-stranded nucleic acid target
sequence to hybridization of known target sequences that have been
previously established, for example, a compilation of melting
curves for known species or a table of digitized data for known
species.
In methods provided herein, one or more single-stranded nucleic
acid target sequences to be analyzed may be provided by nucleic
acid amplification, generally exponential amplification. Any
suitable nucleic amplification method may be used. Preferred
amplification methods are those that generate amplified product
(amplicon) in single-stranded form so that removal of complementary
strands from the single-stranded target sequences to be analyzed is
not required. Probe sets may be included in such amplification
reaction mixtures prior to the start of amplification so that
reaction vessels containing amplified product need not be opened.
When amplification proceeds in the presence of probe sets, it is
preferred that the system be designed such that the probes do not
interfere with amplification. In some embodiments a non-symmetric
PCR method such as asymmetric PCR or, LATE-PCR is utilized to
generate single-stranded copies. PCR amplification may be combined
with reverse transcription to generate amplicons from RNA targets.
For example, reverse transcription may be combined with LATE-PCR to
generate DNA amplicons corresponding to RNA targets or the
complements of RNA targets. In some embodiments, amplification
methods that generate only double-stranded amplicons are not
preferred, because isolation of target sequences in single-stranded
form is required, and melt-curve analysis is more difficult with
double-stranded amplicons due to the tendency of the two amplicons
to collapse and eject hybridization probes. In some embodiments,
methods provided herein do not utilize generation of detectable
signal by digestion of signaling probes, such as occurs in 5'
nuclease amplification assays. In a PCR amplification reaction, for
example, avoidance of probe digestion may be accomplished either by
using probes whose Tm's are below the primer-extension temperature,
by using probes such as those comprising 2' O-methyl
ribonucleotides that resist degradation by DNA polymerases, or by
using DNA polymerases that lack 5' exonuclease activity. Avoidance
of probe interference with amplification reactions is accomplished
by utilizing probes whose Tm's are below the primer-extension
temperature such that the probes are melted off their complementary
sequences during primer extension and, most preferably, during
primer annealing, at least primer annealing after the first few
cycles of amplification. For example, in the amplification assay
method of Example 1, the LATE-PCR amplification method utilized
two-step PCR with a primer-annealing/primer-extension temperature
of 75.degree. C. in the presence of a set of mismatch-tolerant
molecular beacon probes having Tm's against the wild-type target
sequence (to which the probes were perfectly complementary) ranging
from 75.degree. C. to 50.degree. C., which ensured that none of the
probes interfered significantly with amplification of the target
sequence.
In LATE-PCR amplification, for example, the Excess Primer strand is
the single-stranded amplicon to which probe sets hybridize. It
therefore is or contains the single-stranded nucleic acid sequence
that is analyzed. Its 5' end is the Excess Primer, and its 3' end
is the complement of the Limiting Primer. If the sequence to be
analyzed lies between the Excess Primer and the Limiting Primer,
the starting sequence that is amplified and the Excess Primer
strand both contain that sequence. If in the starting sequence to
be amplified the sequence desired to be analyzed includes a portion
of either priming region, it is required that the primer be
perfectly complementary to that portion so that the Excess Primer
strand contain the desired sequence. Primers need not be perfectly
complementary to other portions of the priming regions. Certain
embodiments of methods provide single-stranded nucleic acid target
sequence to be analyzed by amplification reactions that utilize
"consensus primers` that are not perfectly complementary to the
starting sequence to be amplified, and care is taken to ensure that
the Excess Primer strand, which is or contains the single-stranded
target sequence that is actually analyzed, contains the desired
sequence.
Features and embodiments of methods provided herein are illustrated
in the Examples set forth below in conjunction with the
accompanying Figures. All of the Examples illustrate providing as
the single-stranded target sequence or sequences to be analyzed the
Excess Primer strand of a LATE-PCR amplification. Probe sets in the
Examples are designed for contact quenching of signaling probes by
quencher probes.
Example 1 is a case in which a priming region of the
pre-amplification target sequence is included in the sequence
desired to be analyzed. As shown in FIG. 2, eight nucleotides
complementary to the Limiting Primer are included in the sequence
that is to be probed. Example 1 also illustrates the use of a
primer that contains a mismatch. In this case the mismatch lies
outside of the eight nucleotides included in the sequence to be
probed, and the sequence of the Excess Primer strand that is
analyzed is identical to the pre-amplification target sequence 21.
Example 1 illustrates the use of multiple probe sets (three
signaling probes and three quencher probes) to interrogate one
target sequence (a 101-nucleotide long sequence of the rpoB gene of
mycobacterium tuberculosis). The spread of the probes across the
target sequence is shown schematically in FIG. 2. Example 1
illustrates the use of multiple signaling probes of the same color
(all include the fluorophore Quasar 670). Signaling probes 2, 4 and
5 hybridize to different portions of the target sequence and have
different calculated Tm's relative to the wild-type target
sequence--63.degree. C., 67.degree. C. and 75.degree. C.,
respectively. In Example 1 each signaling probe has its own is
associated quencher probe that hybridizes proximate to it, that is,
sufficiently close that its quencher moiety can quench the
signaling probe's fluorophore moiety. Quencher probes 3, 5 and 6
are associated with signaling probes 2, 4 and 5, respectively.
Example 1 illustrates the use of signaling probes that have Tm's
higher than their respective quencher probes. Quencher probes 3, 5
and 6 have Tm's relative to the wild-type target sequence of
50.degree. C., 56.degree. C. and 63.degree. C., respectively, such
that each quencher probe melted off the target sequence before its
associated signaling probe. The two probe sets are not
distinguishable by color, but they are distinguishable be Tm.
Example 1 further illustrates the use of a signaling probe (Probe
2) and a quencher probe (Probe 3) that each has a terminal
nucleotide complementary to the same nucleotide of the
single-stranded nucleic acid target sequence. All six probes were
mismatch-tolerant. The signaling probes and the quencher probes in
this case hybridized adjacently and so covered every nucleotide of
the target sequence that was analyzed. Example 1 illustrates the
use of quenched signaling probes, as each of signaling probes 2, 4
and 5 is a molecular beacon probe with a stem two nucleotides in
length. The example illustrates the use of a quencher probe that
has a hairpin structure (Probe 1) and quencher probes that are
linear probes (Probe 3 and Probe 6). The three probe sets were
tested against a drug-sensitive strain and against two different
drug-resistant strains. Analysis of hybridization of the six probes
against the Excess Primer strand from amplification of the three
strains was by melting. FIGS. 3A-3B present curves showing the
first derivative of fluorescence readings (derivative of the melt
curves). As can be seen from FIGS. 3A-3B, the curve for each
drug-resistant strain differed from the curve for the
drug-sensitive strain and from one another. Thus, the set of probes
was able to determine whether the sample contained the
drug-sensitive sequence or either drug-resistant sequence. Because
each drug-resistant strain had a curve that was distinguishably
different, the set of six probes was able to determine which
drug-resistant strain was present in a sample. The three curves
obtained from these three known sequences could be utilized as a
library against which to compare curves from samples containing
unknown strains.
Example 2 illustrates use of a method provided herein to analyze a
mixture of two starting targets, in this case two variants of a
sequence amplified by a single primer pair. Using starting targets
from Example 1 (the drug-sensitive strain and one drug-resistant
strain), along with the primers and six probes from Example 1,
mixtures of the two strains in proportions varying from 20% to 1%
of the drug-resistant strain were amplified by LATE-PCR to generate
mixtures of two different Excess Primer strands in varying
proportions. As shown in FIGS. 4A-4B, the assay is able to
determine the proportion of drug-resistant strain in the mixtures.
Example 2 also illustrates the use of fluorescence data acquisition
from a melt subsequent to the first melt (in this case the averages
of second, third and fourth melts were used). Experiments conducted
during development of embodiments provided herein demonstrated
that, in some embodiments, the second melt curve differs somewhat
from the first, and that subsequent melt curves agree with the
second. It is contemplated that in some embodiments this is due to
secondary structure that is altered during the first melt, although
the embodiments provided herein are not limited to any particular
mechanism of action and an understanding of the mechanism of action
is not necessary to practice the embodiments. To accommodate the
effect, however, hybridization data, either annealing data or
melting data, can be acquired after an initial melt, which can be a
rapid melt.
Example 3 illustrates an embodiment that includes analysis of three
different variant sequences in the same sample using at least one
probe set for each sequence, wherein probe sets for the three
sequences are detectably distinguishable by color and wherein
different probe sets for one sequence are detectably
distinguishable by Tm. Each variant sequence to be analyzed is
provided by LATE-PCR amplification using an Excess Primer and a
Limiting Primer, and a different primer pair is used for each of
the three variant sequences. As shown in FIG. 5, Example 3
illustrates: the use of one probe (Probe 3) a part of two probe
sets; the use of a probe (quencher Probe 1) that is not part of any
probe set; and probes that hybridize to the single-stranded nucleic
acid target sequence with a gap of one nucleotide between them
(Probe 2 and Probe 3). Probe 2 and Probe 3 are both molecular
beacon probes with a stem two nucleotides long, but whereas none of
the stem nucleotides of Probe 2 is complementary to the target
sequence, two stem nucleotides of Probe 3 are complementary to the
target sequence.
FIGS. 8-11, which accompany Example 4, illustrate the flexibility
of methods provided herein for analyzing sets of signaling probes
and quencher probes that are detectably distinguishable by either
melt or anneal analysis in combination with color. The selected
target sequence is a 500 base-pair portion of the mitochondrial
cytochrome c oxidase subunit 1 gene (cox 1), which overlaps a
sequence that has been used as an identifier of numerous species by
sequencing (Herber, P D (2003) Proc. Bol. Sci. 270 Suppl. 1:S96-9).
Sequences of that gene for 264 different species of nematodes were
aligned and used to identify the selected portion, an area that
contained high-variability sequences flanked by conserved
sequences. In these conserved regions three consensus LATE-PCR
Limiting Primers and a single Excess Primer were designed. The
three consensus Limiting Primers provided sufficient
complementarity to allow amplification of all 264 species above
50.degree. C. The design procedure for all probes was a consensus
sequence that would hybridize to all 264 variants at temperatures
within the range of 30-60.degree. C. The logic of this approach
applies equally to other lengthy variable sequences, for example,
sequences within chloroplasts of plant cells and sequences of
bacteria such as ribosomal genes. FIG. 8 shows the Excess Primer
strand containing a sequence complementary to Limiting Primer Two
for the variant Caenorhabditis elegans with all ten probes
hybridized. Mismatches between each probe and this variant are
identified in Example 4.
Example 4 illustrates the use of an annealing curve to analyze the
probes' hybridization for each color used. FIGS. 9A-9C and FIGS.
10A-10C present annealing curves for the three fluorophores for two
species, including C. elegans. Analysis may include preparation of
a reference file of the annealing curves for all 260 species, and
manual comparison of the curves from an unknown sample with the
curves in the reference file. Experiments were performed during
development of embodiments provided herein to develop a procedure
for digitization of the curves to permit comparison by computer.
FIGS. 11A-11C present the digitized results in graphical, bar code,
form, which can be used for manual comparison to a reference
bar-code file. Example 5 discloses an embodiment of a screening
assay, in this case a sepsis screening assay. Example 5 illustrates
several features and embodiments provided herein. It illustrates
probing and analysis of a variable sequence to determine which
variant is present from among numerous possible variants. It
illustrates the use of multiple sets of signaling and quencher
probes wherein, further, the signaling probes have the same
fluorophore and emit the same color. It illustrates (Table 3) not
only the use of signaling probes whose melting temperatures are
higher against all possible target sequences than their associated
quencher probes, but also inclusion of a set of a signaling probe
and quencher probe where the opposite is the case for one or more
possible target sequences (probes Quasar 1 On and Quasar 1 Off). It
further illustrates the use of multiple probe sets wherein certain
individual probes, signaling or quencher, need not hybridize to
every possible target sequence in the temperature range of
detection, here 80.degree. C. to 25.degree. C. (see Table 3).
Example 5 illustrates analyzing hybridization of signaling and
quencher probes, including the effect of hybridization of quencher
probes on fluorescence emission signals from the signaling probes,
as a function of temperature utilizing annealing curves (FIG. 13)
and derivative curves (FIG. 14), either or both of which can be
maintained as a library against which to compare curves from
unknown samples, and using digitized information derived from such
curves (Table 4), which also can be maintained as a library.
Example 5 illustrates the use of nucleic acid amplification to
provide a sample, or reaction mixture, containing a target sequence
in single-stranded form, in this case a non-symmetric amplification
method that generates a single-stranded amplicon, the target
sequence to be analyzed. Example 5 further illustrates
amplification using a single pair of primers that hybridize to
conserved sequences flanking a variable sequence so as to generate
a target sequence from whichever variant of the variable sequence
is present. It will be appreciated that, as indicated earlier, a
method such as Example 5 or a method such as Example 4 can begin
with a sample containing RNA and include reverse transcription
prior to amplification. Example 5 also illustrates not only
homogeneous detection in which bound signaling probes do not have
to be separated from unbound signaling probes prior to detection,
but also a "single-tube" method in which amplification and
detection are performed without the need to open the reaction
container following amplification. The signaling probes in Example
5 signal upon hybridization to a target, and the probe-target
hybrids have melting temperatures (Table 3) below the amplification
cycling temperatures and, thus, the probes do not hybridize to
amplification products during the amplification reaction. The
probes do not interfere with amplification and are not cleaved
during primer extension by a polymerase having 5' exonuclease
activity, such as Taq DNA polymerase. Probe cleavage would produce
background fluorescence during subsequent melt analysis. Low
temperature probes may be present in the amplification reaction
mixture rather than being added after amplification.
FIGS. 13 and 14 and Table 4 show that the assay of Example 5 is
able to distinguish between variable sequences that differ little
from one another. The assay distinguished staphylococcus epidermis
(SE) from staphylococcus haemolyticus (SH), which differed from one
another at only two nucleotide positions. Thus, as a screening
assay for sepsis, the assay is able to differentiate among
different target sequences not only at the genus level, but also at
the species level.
Example 6 demonstrates the use of the assay of Example 5 with
starting samples that contain mixtures of two variants of the
variable sequence. FIG. 15 presents anneal curves for mixtures of
two variants, staphylococcus aureus (COL) and staphylococcus
epidermis (SE), along with anneal curves for the individual
variants. Starting mixtures of each variant with as little as ten
percent of the other variant were distinguishable from one another
and from the individual variants by the use of a library of curves
or a library of digitized information derived from the curves.
Example 7 extends the Sepsis bacterial detection assay of Example 5
from a 203 base region of the 16S rRNA gene using single color to a
longer 475 base region of 16S rRNA gene using two colors.
Example 8 extends the analysis of the experiment described in
Example 4, using the melting temperatures of the two ON Cal Red
probes and their associated OFF probes, and temperature-dependent
fluorescence signals from those probes in the absence of target
compared to temperature-dependent fluorescence signals from those
probes in the presence of amplified Caenorhabditis elegans target,
presented in FIG. 18. In light of the effective melting
temperatures for Probes 3-6 presented in Example 8, the data in
FIG. 18 and its derivative curve, FIG. 10B, can be explained as
follows. At 65.degree. C. none of the probes is bound to the target
sequence, and the fluorescent signal in both the presence and the
absence of the target sequence is the same. At temperatures below
65.degree. C. the probes hybridize to the target in the order of
their effective melting temperatures: ON Probe 4, OFF Probe 3, OFF
Probe 5, ON Probe 6. Because Probe 4 is a signaling probe it
generates a signal above the background no-target signal when it
binds to the target at about 55.degree. C. As the temperature
decreases below about 52.degree. C. the signal from Probe 4 is
extinguished as quencher Probe 3 binds to the adjacent target
sequence. When the temperature decreases further, OFF Probe 5
hybridizes to the target, but this event is not detected, because
Probe 5 is a quencher probe. When the temperature decreases
further, ON Probe 6 binds to the target adjacent to Probe 5. This
event is detected, because fluorescence coming from unbound
signaling Probe 6 is lost by binding of Probe 6 adjacent to
quencher Probe 5, which is already bound to the target. No signal
above background emanates from signaling Probe 6 when it is
adjacent to quencher Probe 6.
The temperature-dependent signaling generated by hybridization of
the Probe 4/Probe 3 set to the target is independent of the
temperature-dependent signaling generated by hybridization of the
Probe 6/Probe 5 set to the target. It will be appreciated that
Probe 4 and Probe 6 could use chemical moieties that fluoresce in
different colors. It follows that the overall temperature-dependent
fluorescent signal observed in this closed-tube system is comprised
of the integrated signal arising from all independent components of
the system.
The following observation was made from Examples 4 and 8: One or
more sets (or pairs) of interacting probes can be designed in which
the melting temperature of each quencher probe is higher than the
melting temperature of each signaling probe. In this case the
fluorescence emanating from the unbound signaling probes will be
extinguished as each signaling probe binds to the target adjacent
to its already bound quencher probe. When all signaling probes are
bound adjacent to their quencher probes at low temperature, the
system as a whole will display a very low overall fluorescent
signal. Such a system will be very sensitive to the binding or
release of very small amounts of bound signaling probe. The
sensitivity of said system can be increased by using a dabcyl
moiety on the quencher probe, rather than a black hole quencher, or
by having no quencher moiety on the signaling probe. Small amounts
of such probes will hybridize to small amounts of said targets
having an already bound quencher probe. The time required for these
molecules to reach equilibrium between the bound and the unbound
state can be decreased by decreasing the volume of the reaction.
Reactions constructed in this way are amenable to use with
amplification reactions which accumulate small numbers of
single-stranded target molecules rapidly and in small volumes.
Example 9 illustrates the use of a single set of probes for
genotyping of the single nucleotide polymorphism (SNP). The segment
of genomic DNA containing the SNP site to be genotyped was
amplified using LATE-PCR in the presence of the probe set. The ON
probe consisted of a quenched linear probe labeled at the 5' end
with a fluorophore and at the 3' end with a quencher. It was
complementary to both alleles. This probe was designed to have a
melting temperature about 10.degree. C. higher than the OFF Probe
and to hybridize adjacent to the OFF probe binding site such that
upon binding to the LATE-PCR excess primer strand, the fluorophore
moiety of the ON probe resided next the quencher of the OFF probe.
The OFF probe was a linear probe labeled at the 3' end with a
quencher. This probe was designed to be fully matched to one of the
SNP alleles and mismatched to the other allele such that melting
temperature of the OFF probe hybridized to the matched SNP allele
target was about 10.degree. C. higher than its melting temperature
to the mismatched SNP allele target. The relationship of melting
temperatures (Tm's) in the assay was as follows: Limiting Primer
(71.2.degree. C.)>Excess Primer (66.2.degree. C.)>Primer
Annealing (64.degree. C.)>ON Probe (62.degree. C.)>OFF Probe
(52.degree. C. versus matched target, 41.degree. C. versus
mismatched target). As shown in FIG. 20, the fluorescent pattern
generated from this probe pair over a range of detection
temperatures identifies the allele configuration of the SNP site in
the amplified sample: that is, whether the sample is homozygous for
the allele that matches the OFF Probe, whether the sample is
homozygous for the mismatched allele, or whether the sample is
heterozygous and includes both alleles.
Staphylococcus aureus-typing has become an important tool in the
study of strain origin, clonal relatedness, and the epidemiology of
outbreaks. Typing also plays an important role in hospital
investigations, as methicillin-resistant S. aureus (MRSA) is
endemic or epidemic in many institutions. Although several
different phenotypic and, more recently, molecular techniques are
available for differentiating S. aureus, no method is clearly
superior under all conditions. Currently, macrorestriction analysis
by pulsed-field gel electrophoresis (PFGE) is the standard at the
United States of America Centers for Disease Control and Prevention
(CDC) for S. aureus strain typing and has been used successfully to
study strain dissemination, especially in the identification of
nosocomial outbreaks. However, while PFGE has excellent
discriminatory power, it is labor-intensive and difficult to
standardize among different laboratories. As with other gel-based
typing systems, the interpretation of PFGE results is often
subjective. These problems make the exchange of strain typing
information difficult and complicate the creation of an S. aureus
and MRSA typing database.
DNA sequencing is a powerful approach to strain typing with
advantages in speed, unambiguous data interpretation, and
simplicity of large-scale database creation. Recently, DNA
sequencing of the polymorphic X, or short sequence repeat (SSR),
region of the protein A gene (spa) has been proposed as an
alternative technique for the typing of S. aureus. The polymorphic
X region consists of a variable number of 24-bp repeats and is
located immediately upstream of the region encoding the C-terminal
cell wall attachment sequence. The existence of well-conserved
regions flanking the X region coding sequence in spa allows the use
of primers for PCR amplification and direct sequence typing. The
sequencing of the spa SSR region combines many of the advantages of
a sequencing-based system such as MLST but may be more rapid and
convenient for outbreak investigation in the hospital setting,
because spa typing involves a single locus. Inasmuch as the protein
A X region has a high degree of polymorphism, it may have a
variation rate (or clock speed) that provides suitable
discrimination for outbreak investigation." (Shopsin et al., J.
Clinical Microbiology, November 1999, pages 3556-3563)
A different approach to spa typing than PFGE or DNA sequencing
(Shopsin et al) is the use of a LATE-PCR assay using ON/OFF probes
to distinguish strains of S. aureus based on the X repeat region
and to create a signature library where different strains can be
identified. For spa typing there are repeats of 24 bases where each
repeat might have a slightly different sequence and the number of
repeats vary with SPA type. Example 10 describes a LATE-PCR assay
for spa typing utilizing a single set of one signaling (ON) probe
and one quencher (OFF) probe. The ON/OFF probe set was tested
against twelve sequenced spa types of MRSA samples, some of which
had the same spa types, others where spa types were similar, and
still others where the spa type was very different. First
derivative annealing curves of fluorescence versus temperature for
the twelve samples are shown in FIG. 22. All results showed the
expected differentiation and definition of each spa type. When spa
types were expected to be the same, the same signature
appeared.
EXPERIMENTAL
Example 1
Detection of Drug Resistance in the rpoB Gene for Strains of GM.
Tuberculosis
A LATE-PCR amplification was performed using a single pair of
primers to amplify a 150 base pair region of the rpoB gene for each
of several strains of Mycobacterium tuberculosis. The amplification
provided a 101 base-pair region of the gene, which is known to
contain mutations responsible for drug resistance for rifampicin,
as a single-stranded nucleic acid target sequence (the Excess
Primer strand of each LATE-PCR amplification). Following
amplification, each single-stranded nucleic acid target sequence
was probed using six separate probes that were included in the
original amplification reaction mixture.
The probes in combination spanned the 101 base pairs of the
single-stranded nucleic acid target sequence. Three of the probes
were signaling probes. The signaling probes were quenched molecular
beacon probes with two-nucleotide-long stems. Each included
covalently bound labels: the fluorophore Quasar 670 on one end and
a Black Hole Quencher 2, BHQ2, (Biosearch Technologies, Novato,
Calif.), on the other end. The other three probes were quencher
probes terminally labeled with BHQ2 only, with no fluorophore. In
this example the Tm's of the signaling probes with respect to the
drug-sensitive strain differed from one another, and the Tm's of
the quencher probes with respect to the drug-sensitive strain
differed from one another. The three probe sets were detectably
distinguishable.
At the end of amplification, probe-target hybridizations were
analyzed as a function of temperature. In this example,
hybridizations were characterized by the use of melt profile
analysis. Reaction components and conditions were as follows:
TABLE-US-00001 Limiting Primer: (SEQ ID No. 1) 5'
CTCCAGCCAGGCACGCTCACGTGACAGACCG Excess Primer: (SEQ ID No. 2)
5'CCGGTGGTCGCCGCGATCAAGGAG Target: Strain 13545 (SEQ ID No. 3)
5'CCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAG
CCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGCG
CCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGG GCTGGAG Target:
Strain 18460 (SEQ ID No. 4)
5'CCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTG
AGCCAATTCATGGTCCAGAACAACCCGCTGTCGGGGTTGACCCACAAGC
GCCGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCG GGCTGGAG Target:
Strain 9249 (SEQ ID No. 5)
5'CCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTG
AGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCCACAAGC
GCCGACTGTTGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGG GCTGGAG
The underline in the sequence of each of strains 18460 and 9249
denotes the location of the nucleotide change from the
drug-sensitive strain 13545.
TABLE-US-00002 Probe 1: (SEQ ID No. 6)
5'-BHQ2-CTGGTTGGTGCAGAAG-C.sub.3 Probe 2: (SEQ ID No. 7)
5'-BHQ2-TCAGGTCCATGAATTGGCTCAGA-Quasar 670 Probe 3: (SEQ ID No. 8)
5'-BHQ2-CAGCGGGTTGTT-C.sub.3 Probe 4: (SEQ ID No. 9)
5'-BHQ2-ATGCGCTTGTGGATCAACCCCGAT-Quasar 670 Probe 5: (SEQ ID No.
10) 5'-Quasar 670-AAGCCCCAGCGCCGACAGTCGTT BHQ2 Probe 6: (SEQ ID No.
11) 5'-ACAGACCGCCGG BHQ2
A three carbon linker is denoted with C.sub.3 while a Black Hole
Quencher 2 is denoted with BHQ2 (Biosearch Technologies, Novato,
Calif.).
LATE PCR amplifications were carried out in a 25 .mu.l volume
consisting of 1.times. PCR buffer (Invitrogen, Carlsbad, Calif.), 2
mM MgCl2, 200 nM dNTPs, 50 nM Limiting Primer, 1000 nM Excess
Primer, 1.25 units of Platinum Taq DNA Polymerase (Invitrogen,
Carlsbad, Calif.), 500 nM of probes 1, 3 and 6, and 200 nM of
probes 2, 4 and 5. For each strain tested approximately 1000
genomes equivalents were used. Amplification reactions for each
strain were run in triplicate.
The thermal profile for the amplification reaction was as follows:
98.degree. C./3 min for 1 cycle, followed by 98.degree. C./10
s-75.degree. C./40 s for 50 cycles, followed by fluorescent
acquistion at each degree starting with an anneal at 75.degree. C.
with 1.degree. C. decrements at 30 s intervals to 34.degree. C.
followed by 10 min at 34.degree. C. This was followed by a melt
starting at 34.degree. C. with 1.degree. C. increments at 30 s
intervals to 81.degree. C.
The melting temperatures of the probes was performed utilizing the
computer program Visual OMP 7.0 with the concentrations of target,
signaling probes, and quencher probes at 100 nM, 200 nM and 500 nM
respectively. The Tm's were as follows: Probe 1, 50.degree. C.;
Probe 2, 63.degree. C.; Probe 3, 56.degree. C.; Probe 4, 67.degree.
C.; Probe 5, 75.degree. C.; and Probe 6, 63.degree. C. Analysis of
the probe target hybridizations following amplification was by melt
curve analysis using the first derivative for Quasar 670
fluorescence for temperatures between 35.degree. C. to 78.degree.
C. From this data set the highest fluorescent value was used to
normalize the data to one. If the value used was negative, it was
multiplied by (-15); if it was a positive number, it was multiplied
by fifteen.
FIG. 2 illustrates binding of the three prose sets (Probes 1/Probe
2, Probe 3/Probe 4, and Probe 5/Probe 6) to the single-stranded
nucleic acid target sequence utilizing drug-susceptible strain
13545 as the target. In FIG. 2, strand 21 is the target strand,
strand 23 is the Excess Primer, and strand 22 is the Limiting
Primer. For the purpose of illustration probes 1-6 are shown
hybridized to strand 21 in a 3' to 5' orientation with their
mismatched ends above. Mismatches between the probes and strand 21
and between the Limiting Primer and strand 21 are bolded.
Fluorophore and quencher labels are omitted from FIG. 2 but are
given above in the sequence descriptions. Some of the nucleotides
in the probe sequences were deliberately mismatched to the
sensitive strain 13545 such as Probe 1, which contains mismatches
in positions 31(A to G) and 38(T to G) relative to the 5' end of
strand 21. Other mismatches are in Probe 2, position 62(A to A),
Probe 4, position 86 (A to C). Within the Limiting Primer at
position 142(A to G) is a mismatch which was included to reduce a
hairpin that occurred in the original target strand. In addition to
these mismatches in the sensitive strain 13545, strains 18460 has a
nucleotide mismatch at position 59 (T to T) while strain 9249 has a
mismatch at position 104 (G to T).
It will be appreciated that LATE-PCR amplification provides a
sample containing the Excess Primer strand, which comprises the
single-stranded nucleic acid target sequence that is actually
probed. The Excess Primer strand includes the Excess Primer
sequence at one end and the complement of the Limiting Primer
sequence at the other end. In this case, due to the mismatch
between the Limiting Primer and strand 21, the Excess Primer strand
will differ from strand 21 at position 142, which will be a T
rather than a G. As to the region of strand 21 complementary to
probes 1-6, the Excess Primer strand is identical to strand 21.
FIG. 3A presents the results of the analysis for two different
strains of M. tuberculosis, strain 13545 and strain 18460. Data
from analysis of the triplicate samples of the separate
amplifications of the two strains are superimposed for the purpose
of illustration. Circle 311 represents the drug-resistant strain
18460 (D516V, an aspartic acid located at amino acid position 516
changed to a valine), while, circle 312 shows the replicates from
the drug-sensitive strain 13545 (V146F, a valine located at amino
acid position 146 changed to a phenylalanine). FIG. 3B presents the
results for drug-resistant strain 9249 and drug-sensitive strain
13545, where circle 313 shows the replicates for drug-resistant
strain 9249 (S531L, a serine located at amino acid position 513
changed to a leucine) and circle 314 shows the replicates from the
drug-sensitive strain 13545 (V146F).
Example 2
The Detection of a Drug Resistance Strain of M. tuberculosis in a
Mixed Sample
LATE PCR amplifications were performed to provide single-stranded
nucleic acid target sequences using resistant M. tuberculosis
strain 18640 (D516V, an aspartic acid located at amino acid 516
changed to a valine) and the sensitive strain 13545 in different
ratios to determine the level of sensitivity within a mixed sample.
Reaction components and conditions are described in Example 1,
except for the starting target sequences included in the reaction
mixtures. Amplicons generated from strain 18640 and strain 13545
using the primers from Example 1 comprise a single nucleotide
variation within the hybridization sequence of probe 2. In this
embodiment, probe 2 is a signaling probe. Alternatively, in some
embodiments, a quencher probe that hybridizes to the region of the
amplicon containing the variable nucleotide may be employed, and a
corresponding signaling probe is design to hybridize adjacently.
One reaction mixture contained only strain 18460, and another
reaction mixture contained only strain 13545. Each of these 100%
controls contained approximately 100,000 genomic DNA copies of the
pertinent strain. Reaction mixtures for a first mixed sample
contained 20% (approximately 20,000 genomes) of resistant strain
18460 with 80% (approximately 80,000 genomes) of sensitive strain
13545. The reaction mixture for a second mixed sample contained 10%
of strain 18460 (10,000 genomes) with 90% of strain 13545 (90,000
genomes). The reaction mixture for a third mixed sample contained
5% of strain 18460 (5,000 genomes) with 95% of strain 13545 (95,000
genomes). The reaction mixture for a fourth mixed sample contained
1% of strain 18460 (1,000 genomes) with 99% of strain 13545 (99,000
genomes). Amplification reactions were run in triplicate.
The thermal profile for the amplification reaction was as follows:
98.degree. C./3 min for 1 cycle, followed by 98.degree. C./10
s-75.degree. C./40 s for 50 cycles, followed by fluorescent
acquisition at each degree starting with an anneal at 75.degree. C.
with 1.degree. C. decrements at 30 s intervals to 34.degree. C.
then a hold for 10 min at 34.degree. C. This is followed by a melt
starting at 34.degree. C. with 1.degree. C. increments at 30 s
intervals to 81.degree. C. followed by an anneal starting at
75.degree. C. with 1.degree. C. decrements at 30 s intervals to
34.degree. C. This melt/anneal profile was repeated three more
times.
The data used for graphical analysis of the hybridization of the
six probes was the average of each replicate from the last three
melt profiles. From these average values the fluorescence at
35.degree. C. was subtracted, and the resulting values were
normalized by division of all values with the fluorescence at
78.degree. C. The first derivative of the resulting data were then
generated and normalized by dividing all values using the largest
positive value.
In order to remove the contribution of the sensitive strain DNA
from mixtures containing both sensitive and resistant strain DNA's,
replicates of the pure sensitive strain DNA samples (100% controls)
were used to generate average-derived-values at every temperature,
as described above. These values were then subtracted from the
derived-average-values of each mixture to arrive at the
contribution of the resistant strain. In addition, the scatter
among separate samples of pure sensitive DNA was established by
subtracting the derived-average-values of pure sensitive DNA from
each of the individual samples of pure sensitive DNA.
FIGS. 4A-4D show the resulting analysis. They display the signal
from various percentages of the resistant strain 18460 in an
increasing background of sensitive strain 13545. FIG. 4A shows this
signal with a mixed sample of 20% resistant strain 18460 in a
background of 80% sensitive strain 13545, where circle 410
identifies the contribution of the resistant strain in replicates
of the mixture, and circle 411 identifies the scatter among
replicates for the pure sensitive strain. FIG. 4B shows this signal
with the 10% mixture, with circle 412 representing the contribution
of the resistant strain in replicates of the mixture, and circle
413 representing scatter among replicates for the pure sensitive
strain. FIG. 4C shows the signal from the mixture of 5% resistant
strain replicates (circle 414 identifying the contribution of the
resistant strain in replicates of the mixture, and circle 415
identifying scatter among replicates for the pure sensitive
strain). FIG. 4D shows the signal from the mixture of 1% resistant
strain. Circle 416 identifies the contribution of the resistant
strain in replicates of the mixture, and circle 417 identifies the
scatter among replicates of the pure sensitive strain.
Example 3
Multi-drug resistance detection in strains of M. tuberculosis
A multiplex LATE-PCR assay was used to provide multiple
single-stranded target nucleic acids to detect drug resistance in
the three genes, gyrA (fluoroquinolones), katG (isoniazid), and
rpoB (rifampicin), of each of three strains, 13545, 202626 and
15552. For the gyrA gene the strains 13545 and 202626 were
drug-sensitive while strain 15552 (A90V, an aspartic acid located
at amino acid position 90 changed to a valine) was drug-resistant.
For the katG gene the strain 202626 was drug-sensitive, while
strain 13545 (S315T, a serine located at amino acid position 315
changed to a tyrosine) and strain 15552 (S315N, a serine located at
amino acid position 315 changed to a asparagine) were resistant.
For the rpoB gene strain 13545 was a sensitive strain while strain
15552 (S531L, a serine located at amino acid position 513 changed
to a leucine) and strain 202626 (H526D, a histidine located at
amino acid position 513 changed to an aspartic acid) were
resistant.
Reaction components and conditions were as follows:
For the gyrA gene
TABLE-US-00003 Limiting Primer: (SEQ ID No. 12) 5'
ACCAGGGCTGGGCCATGCGCACCA Excess Primer: (SEQ ID No. 13) 5'
GGACCGCAGCCACGCCAAGTC Target: Strain 13545 (SEQ ID No. 14)
5'GGACCGCAGCCACGCCAAGTCGGCCCGGTCGGTTGCCGAGACCATGGG
CAACTACCACCCGCACGGCGACGCGTCGATCTACGACAGCCTGGTGCGCA TGGCCCAGCCCTGGT
Target: Strain 202626 Identical to strain 13545 Target: Strain
15552 (SEQ ID No. 15)
5'GGACCGCAGCCACGCCAAGTCGGCCCGGTCGGTTGCCGAGACCATGGG
CAACTACCACCCGCACGGCGACGTGTCGATCTACGACAGCCTGGTGCGCA TGGCCCAGCCCTGGT
Probe 1: (SEQ ID No. 16) 5' CGACCGGGCC-BHQ2 Probe 2: (SEQ ID No.
17) 5' Cal Red 610-AACCCATGGTCTCGGCAACTT-BHQ2 Probe 3: (SEQ ID No.
18) 5' Cal Red 610-AATCGCCGTGCGGGTGGTAGTT-BHQ2 Probe 4: (SEQ ID No.
19) 5'GCTGTCGTAGATCGACGCG-BHQ2
For the katG gene
TABLE-US-00004 Limiting Primer: (SEQ ID No. 20) 5'
AGCGCCCACTCGTAGCCGTACAGGATCTCGAGGAAAC Excess Primer: (SEQ ID No.
21) 5' TCTTGGGCTGGAAGAGCTCGTATGGCAC Target: Strain 202626 (SEQ ID
No. 22) GCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATC
ACCAGCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACA
ACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCT Target: Strain 13545 (SEQ
ID No. 23) GCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATC
ACCACCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACA
ACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCT Target: Strain 15552 (SEQ
ID No. 24) GCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATC
ACCAACGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACA
ACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCT Probe 1: (SEQ ID No. 25)
5' Cal Orange 560-AAGTGATCGCGTCCTTACCTT-BHQ2 Probe 2: (SEQ ID No.
26) 5' GACCTCGATGCAGCTG-BHQ2
For the rpoB gene Limiting Primer: same as in Example 1 Excess
Primer: same as in Example 1
TABLE-US-00005 Target Strain: 202626 (SEQ ID No. 27) 5'
CCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACCAGCCAGCTGAG
CCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACCGACAAGCGC
CGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGC TGGAG
Target: Strain 15552 Same as strain 9249 set forth in Example 1
Target: Strain 13545 Set forth in Example 1 Probes used for rpoB
gene: Probes 1-6 set forth in Example 1 The underline in a target
sequence denotes the location of the nucleotide change from the
drug sensitive strain.
LATE-PCR amplifications were performed in triplicate carried out in
a 25 ul volume consisting of 1.times.PCR buffer (Invitrogen,
Carlsbad, Calif.), 2 mM MgCl2, 200 nM dNTPs, 50 nM Limiting Primer
and 1000 nM Excess Primer for each primer set, 1.25 units of
Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, Calif.), for the
gyrA probes 500 nM of probes 1 and 3 with 200 nM of probes 2 and 4,
for the katG probes 200 nM of probe 1 and 500 nM of probe 2, and
for the rpoB probes the concentrations set forth in Example 1. For
all strains tested approximately 1000 genomes equivalents of
pre-amplification target were used, and amplification reactions for
each strain were run in triplicate.
The thermal profile for the amplification reaction was as follows:
98.degree. C./3 min for 1 cycle, followed by 98.degree. C./10
s-75.degree. C./40 s for 50 cycles, followed by an anneal starting
at 75.degree. C. with 1.degree. C. decrements at 30 s intervals to
34.degree. C., followed by 10 min at 34.degree. C. This was
followed by a melt starting at 34.degree. C. with 1.degree. C.
increments at 30 s intervals to 81.degree. C.
Probe-target hybridizations were analyzed by the melt curve
analysis using the first derivative for each fluor separately for
the temperatures between 35.degree. C. to 78.degree. C. From each
data set the highest fluorescent value was used to normalize the
data to one. If the value used is negative then it is multiplied by
-15 (minus fifteen), if it was a positive number then it is
multiplied by +15 (plus fifteen). Each of the strains tested
differs in respect to drug resistance. See Table 1 below. For
example, strain 13545 is resistant to isoniazid drugs while
sensitive to both fluorquinolones and rifampicin while strain 15552
is resistant to all three drugs.
TABLE-US-00006 TABLE 1 Drug Gene Strain 13545 Strain 202626 Strain
15552 Fluorquinolones gyrA Sensitive Sensitive Resistant Isoniazid
katG Resistant Sensitive Resistant Rifampicin rpoB Sensitive
Resistant Resistant
FIG. 5 illustrates probe binding of primers and probes to strand
51, the gyrA target of strain 13545, which, because the primers
were perfectly complementary to the original target strand, is
identical to the Excess Primer strand. In FIG. 5 the underlined of
sequence 51 are the nucleotides of the Excess Primer and sequence
52 is the Limiting Primer. Probes 1-4 are shown hybridized to
strand 51 in a 3' to 5' orientation with their unmatched ends
above. The probes are labeled with their respective quenchers or
fluorophores (not shown) as described above. Strain 15552 differs
relative to the 5' end at position 72, a T nucleotide from that of
both strains 13545 and 202626 which has a C nucleotide in that
position.
FIG. 6 illustrates probe binding of primers and probes to strand
61, the katG target of strain 202626, which, because the primers
were perfectly complementary to the original target strand, is
identical to the Excess Primer strand; that is, one of the three
single-stranded products of the LATE-PCR amplification reaction. In
FIG. 6, underlined sequence 63 is the nucleotides of the Excess
Primer, and underlined sequence 62 is the Limiting Primer. Probes
1, 2 are shown hybridized to strand 61 in the 3' to 5' orientation
with their mismatched ends above. Relative to the 5' end of strand
61, all three strains differ at position 56 (G, in bold) to Probe
2. At position 54 is a "G" as shown for strain 202626, but it is a
"C" in strain 13545 and an "A" in strain 15552. The Excess Primer
contains a deliberate mismatch at the 5' end (a "T" rather than the
"G" in each of the targets) to reduce potential mispriming during
the linear phase of LATE-PCR amplification.
The thermal profile for the amplification reaction was as follows:
98.degree. C./3 min for 1 cycle, followed by 98.degree. C./10
s-75.degree. C./40 s for 50 cycles, followed by an anneal starting
at 75.degree. C. with 1.degree. C. decrements at 30 s intervals to
34.degree. C. followed by 10 min at 34.degree. C. This is followed
by a melt starting at 34.degree. C. with 1.degree. C. increments at
30 s intervals to 81.degree. C.
FIG. 7A presents the normalized fluorescence readings of all six
probes for the rpoB gene in three different strains of M.
tuberculosis as a function of the temperature. Circle 711
represents the replicates for strain 202626, while circle 712 shows
the replicates for strain 15552 and circle 713 are the replicates
for strain 13545. FIG. 7B shows the results for the gyrA probes,
which distinguish the sensitive strains 202626 and 13545 (circle
714) from the drug resistant strain 15552 (circle 715). The results
for the katG gene probes are shown in FIG. 7C, in which all three
melt derivatives are different, circle 716 are replicates of the
sensitive strain 202626, while the resistant strains 13545 and
15552 are represented by circle 717 and circle 718,
respectively.
Example 4
Use of Multiple Probes and Multiple Colors for Species-Level
Identification
To demonstrate the ability of embodiments of the methods provided
herein to analyze long sequences, the method was used to
distinguish between nematode species. LATE-PCR assays were
performed using a set of 3 Limiting Primers and an Excess Primer
for the mitochondrial cytochrome oxidase I gene. Reaction
components and conditions were as follows. In the primer and probe
sequences, nucleotides mismatched to the C. elegans sequence are
identified by an asterisk (*). In the probe sequences, nucleotides
added to form a two base-pair stem are underlined.
TABLE-US-00007 Limiting Primer One (SEQ ID No. 28)
5'-GGTT*ATACCTAG*TATAATT*GGTGGTTTTGGTAAT*TG Limiting Primer Two SEQ
ID No. 29) 5'-GGTT*ATACCTAG*TATAATT*GGTGGTTTTGGTAACTG Limiting
Primer Three (SEQ ID No. 30)
5'-GGTT*ATACCTAG*TATAATT*GGTGGTTTTGGC*AAT*TG Excess Primer (SEQ ID
No. 31) 5'-A*CTA*GGATCAAAAAAA*GAAGTATTA*AAATTACGATC Target;
Caenorhabditis elegans (SEQ ID No. 32) 5'-
TCTTGGATCAAAAAATGAAGTATTTAAATTACGATCAGTTAACAACATAG
TAATAGCCCCTGCTAAAACCGGTAGAGATAAAACCAGTAAAAACACTGTT
ACAAATACAGTTCAAACAAATAAAGTTATATGTTCTAATGAAATAGAACT
TCTACGTAAATTTTTAGTAGTACACATAAAATTAATACCACCTAAGATA
GATCTTAACCCTGCTGCATGTAAACTAAAAATAGCTAAATCTACTCTA
CTTCCAGGGTGCCCCATTGTTCTTAAAGGTGGGTAGACTGTTCACCTAG
TCCCACAACCTATATCTACAAAACAAGCATCTAAAATTAATAATATAGAT
GTAGGTAATAACCAAAATCTTAAATTATTTAAACGTGGAAATCTTATA
TCAGGTGCTCCTAACATAAGTGGTAATAATCAGTTACCAAAACCACC GATTATAGTAGGTATTACC
Target; Steinernema feltiae (SEQ ID No. 33) 5'-
TCTAGGATCAAAAAAAGAAGTATTTAAATTACGGTCTGTAAGAAGTATAG
TAATTGCCCCAGCTAAAACCGGTAAAGAAAGAACAAGAAGGAAAACTGT
AACAAAAACAGTTCAAACAAAAAGACTCATATGCTCTAAAGAAATAGAG
CTTCTACGAAGATTCTTAGTAGTACATATAAAATTAATAGCCCCCAAAA
TAGAGCTTACACCAGCACAATGAAGACTAAAAATAGCTAAATCAACCC
TGTTTCCAGGATGGCCTAAAGTACTTAAAGGAGGATAAACAGTTCAAC
TAGTACCACACCCTGTATCTACAAAACAAGCATCTAAAATTAATAATAT
AGCAGTGGGTAATAACCAAAAACTTAAATTATTTAAACGAGGAAATCTT
ATATCCGGAGCACCAAGAAGGAACTAATCAATTTCCAAATCCTCCNNNN NNNNNNNN Probe
Sequences; Probe One (quencher probe for Cal Orange signaling
probe) (SEQ ID N0. 34)
5'-AA*TATTACCT*T*TG*ATGTTAGGG*GCT*CCTGATATAAGT*T TT-BHQ1 Probe Two
(signaling probe with Cal Orange) (SEQ ID No. 35) 5'-CalOrg-
ATCCT*CGTTTAAATAATTTAAGT*TTTTGA*TTATTACCTACT*TCAT- BHQ1 Probe Three
(quencher probe for first Cal Red signaling probe) (SEQ ID No. 36)
5'- TT*TG*TTT*TG*T*T*G*TTG*G*GATT*CTTGTTTTGTT*GATATAGG
TG*GTGGAA-BHQ2 Probe Four (first signaling probe with Cal Red) (SEQ
ID No. 37) 5'-CalRed-A*ACTG*GT*TGAACT*GTT*TACCCT*CCTTTAAGAA
CT*T-BHQ2 Probe Five (quencher probe for second Cal Red signaling
probe) (SEQ ID No. 38) 5'-
AAG*TA*GGT*CAT*CCTGGT*AGTAC*T*GTAGATTTT*GT*TA TTTTTAC*TT-BHQ2 Probe
Six (second signaling probe with Cal Red) (SEQ ID No. 39)
5'-CalRed- ATG*CATGG*T*GCT*GGT*TTT*AGT*TCTATT*TTG*GGTGC*TAT- BHQ2
Probe Seven (quencher probe for first Quasar signaling probe) (SEQ
ID No. 40) 5'- ATTAATTTTATGG*GTACTACTG*T*T*AAG*A*A*T*CT*G*C*G
T*AGTTAT-BHQ2 Probe Eight (first signaling probe with Quasar) (SEQ
ID No. 41) 5'-Quasar-TT CTATTTCT*TTG*GAACATATG*AG*TC*TG*TTTG
TTTGG*ACTGAA-BHQ2 Probe Nine (quencher probe for second Quasar
signaling probe) (SEQ ID No. 42)
5'-TT*TTTGTG*ACT*GTT*TTTTTG*T*TGGTTC*TG*TCTCT*AA- BHQ2 Probe Ten
(second signaling probe with Quasar) (SEQ ID No. 43)
5'-Quasar-TTCCT*GTTTTAGG*T*GGGGCTATTACTATA*TTGTTA ACTAA-BHQ2
FIG. 8 shows strand 81, which is the portion of the Excess Primer
strand that lies between the primers (not shown) from the
amplification of the C elegans target sequence. In FIG. 8, quencher
moieties are shown by (.circle-solid.), Quasar fluorophores are
shown by (*), Cal Red fluorophores are shown by (.star-solid.), and
the Cal Orange fluorophore is shown by (). Oligonucleotides 82, 83,
84, 85, 86, 87, 88, 89, 90 and 91 are Probe Ten through Probe One,
respectively.
The DNA of a single nematode was extracted by placing the
individual worm into 25 ul volume of a lysis buffer containing 100
ug/ml proteinase K, 10 mM Tris-Cl pH 8.3, and 5 uM SDS
(sodium-dodecyl-sulfate); heating to 50.degree. C. for 30 min
followed by 95.degree. C. for 10 min; then adding 25 ul of 10 mM
Tris-C1 pH 8.3 buffer prior to storage at -20.degree. C.
LATE-PCR amplifications were carried out in 25 ul volume consisting
of 1.times. PCR buffer (Invitrogen, Carlsbad, Calif.), 100 nM of
each probe, 3 mM MgCl.sub.2, 250 nM dNTPs, 100 nM of each limiting
primer, 1000 nM of excess primer, 1.25 units of Platinum Taq DNA
polymerase (Invitrogen, Carlsbad, Calif.) and 1 ul of previously
extracted nematode DNA with approximately 10,000 mitochondrial
genomes. Amplification reactions were run in a triplicate sets.
The thermal profile conditions for these reactions were as follows:
95.degree. C. for 3 min followed by 95.degree. C./5 s-55.degree.
C./10 s-72.degree. C./45 s for 5 cycles followed by 95.degree. C./5
s-64.degree. C./10 s-72.degree. C./45 s for 45 cycles followed by a
melt starting at 25.degree. C. with 1.degree. C. increments at 30 s
intervals to 95.degree. C. followed by an annealing starting at
95.degree. C. with 1.degree. C. decrements at 30 s intervals to
25.degree. C. The instrument used for amplification and anneal
analysis was a Bio-Rad IQ5 instrument (Bio-Rad, Hercules,
Calif.).
Probe-target hybridizations were analyzed by anneal curve analysis
using the first derivative for each fluorophore separately (Cal
Orange 560, Cal Red 610 and Quasar 670 from Biosearch Technologies,
Novato Calif.) for temperatures between 65.degree. C. to 25.degree.
C. The fluorescent value at 65.degree. C. is subtracted from all
fluorescent values and thus is zero at 65.degree. C. From this data
set the highest fluorescent value is used to normalize the data to
one. If the value used was negative, it was multiplied by (-15); if
it was a positive number, it was multiplied by fifteen (+15). This
generated numerical values that were subsequently used in a 5-bit
barcoding format.
FIGS. 9A-9C present the normalized fluorescence readings for the
Cal Orange 560, Cal Red 610, and Quasar 670 probes respectively, of
the target Steinernema feltiae as a function of the temperature.
FIG. 9A shows the readings from the Cal Orange 560 probes wherein
circle 911 represents the three replicate amplification reactions.
FIG. 9B shows the readings from the Cal Red 610 probes wherein
circle 912 represents the three replicate amplification reactions.
FIG. 9C shows the readings from the Quasar 670 probes wherein
circle 913 represents the three replicate amplification
reactions.
FIGS. 10A-10C present the normalized fluorescence readings for the
Cal Orange 560, Cal Red 610, and Quasar 670 probes respectively, of
the target Caenorhabditis elegans. FIG. 10A shows the readings from
the Cal Orange 560 probes wherein circle 1011 represents the three
replicate amplification reactions. FIG. 10B shows the readings from
the Cal Red 610 probes wherein circle 1012 represents the three
replicate amplification reactions. FIG. 10C shows the readings from
the Quasar 670 probes wherein circle 1013 represents the three
replicate amplification reactions.
FIGS. 11A-11C show the 5-bit barcoding format that is translated
directly from each of the normalized fluorescent values (Cal Orange
560, Cal Red 610, and Quasar 670 respectively) obtained from the
anneal analysis of the target Steinernema feltiae. The coding is a
5-bit format that represents the fluorescent values obtained at
each one degree decrements in temperature from 65.degree. C. to
25.degree. C. in integer form. For each decrement the first bit is
a determination if the value is either greater or equal to zero,
which is scored as plus (black color) while values below zero are
scored as a minus (no color). The next 4 bits represent the
integers 1, 2, 4, and 8 for the fluorescent values obtained by the
analysis. For example, if at temperature 45.degree. C. the integer
value from the Cal Orange 560 fluorescence is 12, then bit 1 has no
color, bits 2 and 3 (representing intergers 1 and 2) have no color,
and bits 4 and 5 (representing intergers 4 and 8) are black. FIG.
11A is the fluorescence values obtained from the Cal Orange 560
probes converted into a 5-bit barcode from a single reaction and
shows how the barcode is arranged at each temperature with the bits
arranged vertically. FIG. 11B is the fluorescence values obtained
from the Cal Red 610 probes converted into a 5-bit barcode from a
single reaction. FIG. 11C is the fluorescence values obtained from
the Quasar 670 probes converted into a barcode from a single
reaction.
Example 5
Sepsis Assay
Sepsis may result from infection by any of a number of bacterial
species. The assay presented in this example demonstrates the
ability to distinguish among species using the analytical methods
provided herein with a single-tube, homogeneous LATE-PCR
amplification and detection method. A region of the bacterial 16s
ribosomal gene was chosen for analysis, because the region is known
to have relatively conserved sequences that flank a hypervariable
region. A single Limiting Primer and a single Excess Primer
complementary to conserved sequences flanking the V3 hypervariable
region of the 16s gene wee used.
For a test panel, eleven bacterial species identified in Table 2
were utilized (abbreviations used throughout the remainder of the
example are provided in Table 2).
TABLE-US-00008 TABLE 2 Bacterial Species Panel Bacterial Species
Abbreviation Acinetobacter baumannii AB Acinetobacter sp. ASP
Enterobacter aerogenes EA Enterobacter cloacae EC Enterococcus
faecalis ENFS Enterococcus faecium ENFM Klebsiella pneumoniae KP
Pseudomonas aeruginosa PA Staphylococcus aureus COL Staphylococcus
epidermidis SE Staphylococcus haemolyticus SH
The gene sequence to be utilized was selected by examining the
sequences of the species in the panel for a sequence that fits the
criteria described above and whose variable region includes
sufficient differences among the target species and closely related
non-target species by means of a BLAST search, a software program
that compares sequences to a known library of sequences, comparison
in the NCBI Genbank, a known United States national library
sequence database. By this method, which is known in the art, a 203
base pair region was selected, namely, nucleotides 325-527, of
Klebsiella pneumoniae, NCBI Genbank reference number NC_011283 of
16s rRNA. The gene sequence for the above region of Klebsiella
pneumoniae was downloaded into a computer program for primer and
probe design. Visual OMP (DNASoftware, Inc., Ann Arbor, Mich., USA)
was used for the assay design software. Using the design software,
a primer set and a set of probes comprising four signaling probes
(we refer to them for convenience as "ON" probes) and four quencher
probes (we refer to them for convenience as "OFF" probes) was
designed. To reach a final design of primers and probes, the
initial design was treated as prospective. Several of the sequences
selected as design sequences were run through another BLAST search
to confirm the appropriate homology with the target sequences and
to confirm that the primers have sufficient difference from
non-target organisms to avoid their amplification/detection. Next
one target species of Table 2, Staphylococcus aureus, was tested in
a separate amplification utilizing bacterial genomic DNA with the
primers and with SYBR Green dye for detection using real-time PCR
and melt-curve analysis to check for acceptable amplification
efficiency as determined by the linearity of threshold cycle
(C.sub.T) as a function of target concentration and production of a
specific amplification product ("amplicon") as measured by
melt-curve analysis.
Using the foregoing method, the following primers and probes were
designed. It will be noted that each of the signaling, or "ON",
probes is a molecular beacon probe having a stem of two
nucleotides, with addition of nucleotides that are not
complementary to the target sequences as needed (such added
nucleotides being bolded for identification). It will be noted also
that all of the signaling probes have a Quasar 670 fluorophore
(Biosearch Technologies, Novato, Calif., USA) on one end and a
Black Hole Quencher 2 ("BHQ2") quencher (Biosearch Technologies) on
the other end, whereas all the quencher probes have a Black Hole
Quencher 2 but no fluorophore. The stated primer and probe Tm's are
the calculated concentration-adjusted melting temperatures used for
LATE-PCR.
TABLE-US-00009 Primer Pair Limiting Primer: (SEQ ID No. 44)
CCAGACTCCTACGGGAGGCAGCAGT, Tm = 74.7 Excess Primer: (SEQ ID No. 45)
GTATTACCGCGGCTGCTGGCA, Tm = 72.1 Probe "Quasar con 1 off": (SEQ ID
No. 46) AAGGGGAATATTGCACAATGGTT-BHQ2 Probe "Quasar con 1 on": (SEQ
ID No. 47) Quasar 670-AAGCGAAAGCCTGATGCAGCCATT-BHQ2 Probe "Quasar
con 2 on": (SEQ ID No. 48) BHQ2-TAGCCGCGTGTGTGAAGAATA-Quasar 670
Probe "Quasar con 2 off": (SEQ ID No. 49)
BHQ2-TTGGCCTTCGGATTGTAAAGCACTTAA-C3 Carbon Linker Probe "Quasar 1
off": (SEQ ID No. 50) TATTAGTAGGGAGGAAGTA-BHQ2 Probe "Quasar 1 on":
(SEQ ID No. 51) Quasar 670-TTATATGTGTAAGTAACTGTGCACATCAA-BHQ2 Probe
"Quasar 2 off": (SEQ ID No. 52) TTGACGTTACCCGCAA-BHQ2 Probe "Quasar
2 on": (SEQ ID No. 53) Quasar
670-TTGAAGAAGCACCGGCTAACTCCGAA-BHQ2
The alignment of the primers and probes on the target sequences
selected as design sequences is shown in FIG. 12, which presents
one strand only of each target sequence. Nucleotide positions are
shown in the right-hand column of FIG. 12. Sequences in the column
designated 121 correspond to the Limiting Primer, and sequences 130
correspond to the Excess Primer. The location of quencher probe
"Quasar con 1 off" is the column of sequences 122. The location of
signaling probe "Quasar con 1 on" is sequences 123. The location of
signaling probe "Quasar con 2 on" is sequences 124. The location of
quencher probe "Quasar con 2 off" is sequences 125. The location of
quencher probe "Quasar 1 off" is sequences 126. The location of
signaling probe "Quasar 1 on" is sequences 127. The location of
quencher probe "Quasar 2 off" is sequences 128. The location of
signaling probe "Quasar 2 on" is sequences 129.
The melting temperatures (Tm's) of the quencher probes (300 nM) and
loop portions of the signaling probes (100 nM) in the probe set
against the various design target sequences (FIG. 12) that are
representative of the clinical bacterial species found in sepsis,
as predicted by the Visual Omp design program, are shown in Table
3.
TABLE-US-00010 TABLE 3 Calculated Probe Melting Temperatures,
.degree. C. PROBE Quasar Quasar Quasar Quasar Sequence Con1 Off
Con1 On Con 2 On Con 2 Off KP 60.3 67.1 62.8 63.4 EA 60.3 67.1 56.1
54.2 AB 50.5 51.7 62.8 44.1 PA 50.5 65.6 62.8 59.9 COL 13.1 50.2
49.6 26.8 SE 2.2 50.2 49.6 26.8 ENFS 6.4 23.8 54.0 15.2 PROBE
Sequence Quasar 1 Off Quasar 1 On Quasar 2 Off Quasar 2 On KP 35.5
13.0 54.4 65.5 EA 20.2 13.0 40.0 65.5 AB 20.2 19.5 38.1 60.5 PA
36.2 -7.4 39.7 57.9 COL 20.2 62.4 8.4 34.8 SE 20.2 53.4 8.4 34.8
ENFS 20.2 21.7 -2.4 34.8
Twenty-five .mu.L LATE-PCR reaction mixtures including a single
bacterial genomic DNA target contained 10.times.PCR Buffer 1.times.
(final concentration), 10 mM dNTPs 250 .mu.M (final concentration),
50 mM Mg.sup.++3 mM (final concentration), 10 .mu.M Limiting Primer
50 nM (final concentration), 100 .mu.M Excess Primer 1000 nM (final
concentration), 10 .mu.M each signaling probe 100 nM (final
concentration), 10 .mu.M of each quencher probe 300 nM (final
concentration), 1 Unit Platinum Taq DNA polymerase and 10.sup.6
bacterial genomic DNA starting copies. Two controls were also
amplified: a probes-only control (NTCP) containing all above
reagents except Taq polymerase and genomic DNA, and a Taq
polymerase-only control (NTCT) containing Taq polymerase and all of
the above reagents but no genomic DNA.
Amplification and detection of three replicate samples of each
target and each control were performed with a Bio Rad (Hercules,
Calif., USA) IQ5 real-time thermocycler using the following
protocol: denaturation at 95.degree. C. for three minutes, followed
by 35 cycles of 95.degree. C. for 10 seconds, 69.degree. C. for 15
seconds and 72.degree. C. for 45 seconds. Following amplification a
melt curve was generated starting at 25.degree. C. and progressing
to 80.degree. C. in one-degree steps of thirty seconds each and
then an anneal curve was generated starting at 80.degree. C. and
progressing to 25.degree. C. in one degree steps of thirty seconds
each, with data acquisition of Quasar 670 fluorescence at each
step. The various anneal curves are shown in FIG. 13, a graph of
the normalized intensities of Quasar 670 fluorescences versus
temperature. Fluorescence intensities were normalized to 75.degree.
C. and with background fluorescence of the NTCT control at 75 deg
subtracted, and then the highest fluorescent value normalized to
1.0. In FIG. 13, circle 131 is the anneal curves for the NTCP
control, circle 132 is the anneal curves for the NTCT control,
circle 133 is the anneal curves for target AB, circle 134 is the
anneal curves for the target ASP, circle 135 is the anneal curves
for the target EA, circle 136 is the anneal curves for target EC,
circle 137 is the anneal curves for target ENFS and for target ENFM
which should be the same, circle 138 is the anneal curves for
target KP, circle 139 is the anneal curves for the target PA,
circle 140 is the anneal curves for the target COL, circle 141 is
the anneal curves for the target SE, and circle 142 is the anneal
curves for the target SH.
FIG. 14 presents the anneal derivative, -dF/dT of the anneal
curves. The numbered circles in FIG. 14 identify the derivative
curves as follows: circle 151 is the three replicates of the NTCP
control; circle 152, for the NTCT control; circle 153, for target
AB; circle 154, for target ASP; circle 155, for target EA; circle
156, for target EC; circle 157, for target ENFS and target ENFM
that give the same signal; circle 158, for target KP; circle 159,
for target PA; circle 160, for target COL; circle 161, for target
SE; and circle 162, for target SH.
The fluorescence intensity curves (FIG. 13) or the derivative
curves (FIG. 14) can be used as a library. A curve or curves from
an unknown sample can be compared to the stored curve or curves to
identify the bacterial species that is present. Alternatively or in
addition, digitized information from either or both families of
curves can be used as a library. For example, for each replicate of
each target in FIG. 13, one can create a table of ratios. One such
table constructed during development of embodiments of the present
invention is the ratio of fluorescence intensity at 25.degree. C.
to the intensity at 30.degree. C., the ratio of intensity at
30.degree. C. to the intensity at 35.degree. C. and so on up the
temperature scale five degrees at a time. Ratios resulting from an
unknown sample can be compared to the library of ratios to identify
the species that is present. Alternatively, from FIG. 14 one can
prepare a table of temperatures at which maxima and minima occur.
Table 4 presents such temperatures for the curves of FIG. 14, where
a positive (+) indicator represents the temperature of a maximum
and a negative (-) indicator represents the temperature of a
minimum. Maxima and minima from an unknown can be compared to Table
4 to identify the species present.
TABLE-US-00011 TABLE 4 Maximum and Minimum Temperature Values of
Anneal Derivatives Bacterium Temperatures (.degree. C.) of Maxima
(+) and Minima (-) AB (+) 61, (-) 55, (+) 52, (-) 46 ASP (+) 60,
(-) 53 EA (+) 66, (-) 63, (+) 60, (-) 54, (+) 52, (-) 49 EC (+) 68,
(-) 62, (+) 59, (-) 56 ENFS (+) 55, (-) 36 ENFM (+) 55, (-) 36 KP
(+) 68, (-) 63 PA (+) 65, (-) 61, (+) 58, (-) 52, (+) 47, (-) 41
COL (+) 61, (-) 55, (+) 50, (-) 43, (+) 37 SE (+) 50, (-) 43, (+)
37 SH (+) 49, (-) 43, (+) 37
Table 4 shows that the Tms for most of the samples are very
different because their 16S rRNA region is highly variable, so even
those species of the same genus like EA and EC, and AB and ASP have
very different values in Table 4. However for ENFS and ENFM that
have the same 16S DNA sequence, the values are the same, as is
predicted. Even those targets with as few as two DNA sequence
differences, SE and SH, show a different set of values in Table 4,
and have significant differences versus COL.
Example 6
Sepsis Assay with More than a Single Bacterial Species Present
In clinical situations more than a single bacterial species may be
present and the Sepsis Assay must be able to differentiate between
a fluorescent signal pattern generated from a single bacteria
species to that from a mixture of two or more species.
Using the same primers and probes, and PCR reagents as in Example
5, except that two bacterial genomic DNA targets are included, FIG.
15 shows the anneal curves after normalization of the raw data to
75.degree. C., the NTCT background subtracted at 75.degree. C., and
the highest fluorescent value normalized to 1.0 for different
mixtures of two bacteria species, where the starting concentration
of one species is held constant while the starting concentration of
the second species is varied. Circle 170 are the NTCT; circle 171
are 10.sup.5 starting copies of only SE; circle 172 are 10.sup.6
starting copies of only COL; circle 173 are a mixture of 10.sup.6
starting copies of COL and 10.sup.5 starting copies of SE; circle
174 are a mixture of 10.sup.5 starting copies of COL and 10.sup.5
starting copies of SE, circle 175 are a mixture of 10.sup.4
starting copies of COL and 10.sup.5 starting copies of SE.
FIG. 15 shows the resolution of fluorescent signatures for
mixtures. A specific fluorescent signature is given for both the
pure samples and each of the mixtures. In FIG. 15, mixtures of
fluorescent signatures of COL: SE of 1.0:0.0, 1.0:0.9, 1.0:1.0,
0.9:1.0 and 0.0:1.0 are shown. The fluorescence intensity curves
(FIG. 15) can be used as a library. An unknown sample can be
compared to the stored curve or curves to identify the bacterial
specie or mixture of species that are present. Alternatively or in
addition, digitized information from families of curves can be used
as a library. For example, for each replicate of each target in
FIG. 15, one can create a table of ratios. One such table is the
ratio of fluorescence intensity at 25.degree. C. to the intensity
at 30.degree. C., the ratio of intensity at 30.degree. C. to the
intensity at 35.degree. C. and so on up the temperature scale five
degrees at a time. Ratios resulting from an unknown sample can be
compared to the library of ratios to identify the species or
combination of species that is present. From these data it is also
evident that mixtures where the minor component is less than 10% of
the major component will not be resolved. The library of
fluorescent signatures can be developed for all mixtures between
relative concentrations of 1.0 to 0.1 of each component taken in
0.1 steps.
Example 7
Sepsis Assay
The gene sequence to be utilized was selected by examining the
sequences of the species in the panel shown in Table 2 in Example 5
for a sequence that fits the criteria described above and whose
variable region includes sufficient differences among the target
species and closely related non-target species by means of a BLAST
search, a software program that compares sequences to a known
library of sequences, comparison in the NCBI Genbank, a known
United States national library sequence database. By this method, a
475 base pair region was selected, namely, nucleotides 321-795, of
Acinetobacter baumannii (AB), NCBI Genbank reference number
NC_010400 of 16s rRNA. The gene sequence (see FIG. 16) for the
above region of AB was downloaded into a computer program for
primer and probe design. Visual OMP (DNASoftware, Inc., Ann Arbor,
Mich., USA) was used for the assay design software. Using the
design software, a primer set and a set of probes was designed
comprising eight signaling ("ON") probes and eight quencher ("OFF")
probes. To reach a final design of primers and probes, the initial
design was treated as prospective. Several of the sequences were
selected as design sequences, which were run through another BLAST
search to confirm the appropriate homology with the target
sequences and to confirm that the primers have sufficient
difference from non-target organisms to avoid their
amplification/detection. Next one target species, AB, was tested in
a separate amplification utilizing bacterial genomic DNA with the
primers and with SYBR Green dye for detection using real-time PCR
and melt-curve analysis to check for acceptable amplification
efficiency as determined by the linearity of threshold cycle
(C.sub.T) as a function of target concentration and production of a
specific amplification product ("amplicon") as measured by
melt-curve analysis.
Using the foregoing method, the following primers and probes were
designed. Each of the signaling, or "ON", probes is a molecular
beacon probe having a stem of two nucleotides, with addition of
nucleotides that are not complementary to the target sequences as
needed (such added nucleotides being bolded in the sequences for
identification). Four of the signaling probes have a Cal Red 610
fluorophore (Biosearch Technologies, Novato, Calif., USA) on one
end and a Black Hole Quencher 2 ("BHQ2") quencher (Biosearch
Technologies) on the other end, and four of the signaling probes
have a Quasar 670 fluorophore (Biosearch Technologies, Novato,
Calif., USA) on one end and a BHQ2 quencher on the other end. All
eight of the quencher probes have a BHQ2 quencher but no
fluorophore. The stated primer and probe Tm's are the calculated
concentration-adjusted melting temperatures used for LATE-PCR.
TABLE-US-00012 Primer Pair Limiting Primer: (SEQ ID No. 54)
CCAGACTCCTACGGGAGGCAGCAGT, Tm = 74.7 Excess Primer: (SEQ ID No. 55)
TGGACTACCAGGGTATCTAATCCTGTTTG, Tm = 69.2 Probe "Cal Red 5 off":
(SEQ ID No. 56) ATAGGGTGCGAGCGTTAATCT-BHQ2 Probe "Cal Red 5 on":
(SEQ ID No. 57) Cal Red 610-AAGGATTTACTGGGCGTAAAGCGTT- BHQ2 Probe
"Cal Red 6 off": (SEQ ID No. 58) TTGCGTAGGCGGCTTATTAAGTAA-BHQ2
Probe "Cal Red 6on": (SEQ ID No. 59) Cal Red
610-AACGGATGTGAAATCCCCGAGCTT-BHQ2 Probe "Cal Red 7off": (SEQ ID No.
60) TAACTTGGGAATTGCATTCGTA-BHQ2 Probe "Cal Red 7on": (SEQ ID No.
61) Cal Red 610-ATACTGGTGAGCTAGAGTATGAT-BHQ2 Probe "Cal Red 8off":
(SEQ ID No. 62) GAAGAGGATGGTAGAATTCC-BHQ2 Probe "Cal Red 8on": (SEQ
ID No. 63) Cal Red 610-TAGGTGTAGCGGTGAAATGCGTA-BHQ2 Probe "Quasar
con 1 off": (SEQ ID No. 64) AAGGGGAATATTGCACAATGGTT-BHQ2 Probe
"Quasar con 1 on": (SEQ ID No. 65) Quasar
670-AAGCGAAAGCCTGATGCAGCCATT-BHQ2 Probe "Quasar con 2 on": (SEQ ID
No. 66) BHQ2-TAGCCGCGTGTGTGAAGAATA-Quasar 670 Probe "Quasar con 2
off": (SEQ ID No. 67) BHQ2-TTGGCCTTCGGATTGTAAAGCACTTAA-C3 Carbon
Linker Probe "Quasar 1 off": (SEQ ID No. 68)
TATTAGTAGGGAGGAAGTA-BHQ2 Probe "Quasar 1 on": (SEQ ID No. 69)
Quasar 670-TTATATGTGTAAGTAACTGTGCACATCAA-BHQ2 Probe "Quasar 2 off":
(SEQ ID No. 70) TTGACGTTACCCGCAA-BHQ2 Probe "Quasar 2 on": (SEQ ID
No. 71) Quasar 670-TTGAAGAAGCACCGGCTAACTCCGAA-BHQ2
The alignment of the primers and probes on the target sequences
selected as design sequences is shown in FIG. 16, which presents
one strand only of the AB target sequence. Nucleotide positions are
shown in the left-hand column of FIG. 16. Sequence 176 corresponds
to the Limiting Primer, and sequence 193 corresponds to the reverse
compliments of the Excess Primer. The location of quencher probe
"Quasar con 1 off" is sequence 177. The location of signaling probe
"Quasar con 1 on" is sequence 178. The location of signaling probe
"Quasar con 2 on" is sequence 179. The location of quencher probe
"Quasar con 2 off" is sequence 180. The location of quencher probe
"Quasar 1 off" is sequence 181. The location of signaling probe
"Quasar 1 on" is sequence 182. The location of quencher probe
"Quasar 2 off" is sequence 183. The location of signaling probe
"Quasar 2 on" is sequence 184. The location of quencher probe "Cal
Red 5 off" is sequence 185. The location of signaling probe "Cal
Red 5 on" is sequence 186. The location of quencher probe "Cal Red
6 off" is sequence 187. The location of signaling probe "Cal Red 6
on" is sequence 188. The location of quencher probe "Cal Red 7 off"
is sequence 189. The location of signaling probe "Cal Red 7 on" is
sequence 190. The location of quencher probe "Cal Red 8 off" is
sequence 191. The location of signaling probe "Cal Red 8 on" is
sequence 192.
The melting temperatures (Tm's) of the quencher probes (300 nM) and
loop portions of the signaling probes (100 nM) in the probe set
against the various design target sequences (FIG. 16) that are
representative of the clinical bacterial species found in sepsis,
as predicted by the Visual Omp design program, are shown in Table
5
TABLE-US-00013 TABLE 5 Calculated Probe Melting Temperatures,
.degree. C. PROBE Quasar Quasar Quasar Quasar Sequence Con1 Off
Con1 On Con 2 On Con 2 Off KP 60.3 67.1 62.8 63.4 EA 60.3 67.1 56.1
54.2 AB 50.5 51.7 62.8 44.1 PA 50.5 65.6 62.8 59.9 COL 13.1 50.2
49.6 26.8 SE 2.2 50.2 49.6 26.8 ENFS 6.4 23.8 54.0 15.2 PROBE
Sequence Quasar 1 Off Quasar 1 On Quasar 2 Off Quasar 2 On KP 35.5
13.0 54.4 65.5 EA 20.2 13.0 40.0 65.5 AB 20.2 19.5 38.1 60.5 PA
36.2 -7.4 39.7 57.9 COL 20.2 62.4 8.4 34.8 SE 20.2 53.4 8.4 34.8
ENFS 20.2 21.7 -2.4 34.8 PROBE Cal Red Sequence Cal Red 5 Off Cal
Red 5 On Cal Red 6 Off 6 On KP 56.6 60.5 46 64.2 EA 56.6 60.5 46
64.2 AB 62.6 64.4 65.2 69.5 PA 56.4 60.5 32.7 60.4 COL 26.8 55.6
52.5 8.7 SE 29.4 55.6 52.5 8.7 ENFS 37.2 55.4 49.4 41.7 PROBE Cal
Red Sequence Cal Red 7 Off Cal Red 7 On Cal Red 8 Off 8 On KP 48.2
24.7 37.2 67.1 EA 48.2 24.7 37.2 67.1 AB 59.1 59.4 55.9 67.1 PA
36.1 37.2 15.7 64 COL -5.2 12.8 23.1 63.6 SE -5.2 12.8 23.1 63.6
ENFS -3.1 12.8 15.5 64
Twenty-five .mu.L LATE-PCR reaction mixtures including a single
bacterial genomic DNA target contained 10.times.PCR Buffer 1.times.
(final concentration), 10 mM dNTPs 250 .mu.M (final concentration),
50 mM Mg.sup.++3 mM (final concentration), 10 .mu.M Limiting Primer
50 nM (final concentration), 100 .mu.M Excess Primer 1000 nM (final
concentration), 10 .mu.M each signaling probe 100 nM (final
concentration), 10 .mu.M of each quencher probe 300 nM (final
concentration), 1 Unit GE puRe Taq DNA polymerase, 0.5 bead, and
10.sup.6 bacterial genomic DNA starting copies. A Taq
polymerase-only control (NTC) containing Taq polymerase and all of
the above reagents but no genomic DNA was also amplified.
Amplification and detection of three replicate samples of each
target and each control were performed with a Bio Rad (Hercules,
Calif., USA) IQ5 real-time thermocycler using the following
protocol: denaturation at 95.degree. C. for three minutes, followed
by 50 cycles of 95.degree. C. for 10 seconds, 65.degree. C. for 15
seconds and 72.degree. C. for 45 seconds. Following amplification a
melt curve was generated starting at 25.degree. C. and progressing
to 80.degree. C. in one-degree steps of thirty seconds each and
then an anneal curve was generated starting at 80.degree. C. and
progressing to 25.degree. C. in one degree steps of thirty seconds
each, with data acquisition of Cal Red 610 and Quasar 670
fluorescence at each step. The various anneal curves are shown in
FIG. 17A, a graph of the normalized intensities of Cal Red 610
fluorescence versus temperature, and FIG. 17B, a graph of the
normalized intensities of Quasar 670 fluorescence versus
temperature. Fluorescence intensities were normalized to 75.degree.
C. and with background fluorescence of the NTC control at 75 deg
subtracted, and then the highest fluorescent value normalized to
1.0. In FIG. 17A, circle 194 is the anneal curves for target AB,
circle 195 is the anneal curves for the target ASP, circle 196 is
the anneal curves for the target EA and for target KP which should
be the same, circle 197 is the anneal curves for target ENFS and
for target ENFM which should be the same, circle 198 is the anneal
curves for the target PA, circle 199 is the anneal curves for the
target COL, for the target SE, and for the target SH which should
be the same. In FIG. 17B, circle 200 is the anneal curves for
target AB, circle 201 is the anneal curves for the target ASP,
circle 202 is the anneal curves for the target EA, circle 203 is
the anneal curves for target ENFS and for target ENFM which should
be the same, circle 204 is the anneal curves for target KP, circle
205 is the anneal curves for the target PA, circle 206 is the
anneal curves for the target COL, circle 207 is the anneal curves
for the target SE, and circle 208 is the anneal curves for the
target SH.
One could analyze the data in FIGS. 17A and 17B by preparing
derivative curves and comparing maximum and minimum temperature
values, as described in example 5 and shown in Table 4.
Example 8
Further Analysis of the Experiment of Example 4
Using the sequence information presented in Example 4 and a
computer program known as VISUAL OMP version 7.2.0.0 the effective
melting temperature was determined for the two Cal Red signaling
probes and their associated quencher probes to their C. elegans
target sequences. The results were as follows: Probe 3 (OFF):
51.7.degree. C., Probe 4 (ON): 53.3.degree. C., Probe 5 (OFF):
41.2.degree. C., Probe 6 (ON): 26.3.degree. C.
Probe 3 and Probe 4 together comprise a set (or pair) of
interacting probes, and Probe 5 and Probe 6 together comprise a
separate set (or pair) of interacting probes. The difference in the
melting temperatures of Probe 3 and Probe 4 is =(+)1.6.degree. C.,
while the difference in the melting temperatures of Probe 5 and
Probe 6 is =(-) 15.9.degree. C. In the Probe 3/Probe 4 set the
signaling probe has the higher calculated melting temperature,
whereas in the Probe 5/Probe set the quencher probe has the higher
calculated melting temperature.
FIG. 10B presents the normalized derivative curve of fluorescence
readings for Cal Red 610 probes of the target Caenorhabditis
elegans, wherein circle 1012 represents the three replicate
amplification reactions. The conditions, the target and
experimental details for the data in FIG. 10B are described in
Example 4. FIG. 18, circle 218, identifies the
temperature-dependent composite fluorescent signals of the anneal
curves in this reaction. These data were used to generate the
temperature-dependent first derivative curves shown in FIG. 10B.
FIG. 18, circle 219, identifies the temperature-dependent composite
fluorescent signals of Probes 3-6 in the absence of a template for
amplification. Comparison of the fluorescent signals in FIG. 18
reveals that the signal in the presence of amplified target (circle
218) has a higher value than the signal in the absence of target
(signal 219) in the range of 53.degree. C., it has a lower value at
temperatures approximately between 52 and 25.degree. C.
When the melting temperature of a quencher probe is higher than the
melting temperature of the signaling probe, as the temperature is
decreased, binding of the signaling probe to the target is detected
as a decrease in fluorescence. In order to maximize the decrease in
fluorescence due to binding of a signaling probe, the concentration
of quencher probe molecules should at least equal, and preferably
exceed, the concentration of signaling probe molecules, and the
melting temperature of the quencher probe to the target (any target
of known sequence) should preferably at least 5.degree. C. greater,
and most preferably at least 10.degree. C. greater, than the
melting temperature of the signaling probe. Under these conditions
the magnitude of the temperature-dependent decrease in fluorescence
will depend on the concentration of target molecules present in the
reaction, as illustrated in FIG. 19.
When no target molecules are present in the reaction, FIG. 19, line
220, the extent of the temperature-dependent decrease in
fluorescence in the system will depend on the chemical composition
of the signaling probe oligonucleotide, including: 1) its length;
2) the nature of the covalently linked fluorescent moiety; 3) the
presence or absence of a quencher moiety on the oligonucleotide; 4)
the nature of an covalently linked fluorescent moiety. The
temperature-dependent decrease in fluorescence will reach a maximum
when the concentration of target molecules present in the reaction
exceeds the concentration of signaling molecules present in the
reaction, illustrated in FIG. 19 by line 222, provided the
concentration of target molecules does not exceed the concentration
of quencher molecules. What will be seen at intermediate
concentrations of target molecules is illustrated in FIG. 19 by
line 221: the magnitude of the decrease in fluorescence will be
intermediate the maximum possible fluorescence, FIG. 19, line 222,
and the minimum possible fluorescence, FIG. 19 line 220. By knowing
the concentration of signaling probes present in a reaction, a
person skilled in the art could use this approach to establish the
concentration of target molecules present in a reaction at any
time, including: a) in the presence of absence of target
amplification; b) during target amplification; and/or c) after
target amplification.
Example 9
SNP Genotyping
This example illustrates the use of probes for genotyping of the
single nucleotide polymorphism (SNP) rs373129 located in the human
tumor suppressor gene CDKN2A.
The segment of genomic DNA containing the SNP site to be genotyped
was amplified using LATE-PCR. The probes were designed to hybridize
at temperatures 10.degree. C. below the melting temperature of the
limiting primer used for LATE-PCR amplification. Tm calculations
were performed using the Visual OMP software from DNA software (Ann
Arbor, Mich.). The OFF probe consisted of a linear probe labeled at
the 3' end with a Black Hole Quencher 2 (BHQ2), Biosearch
Technologies, Inc., Novato Calif. This probe was designed to be
fully matched to one of the SNP alleles and mismatched to the other
allele such that calculated melting temperature of the OFF probe
hybridized to the matched T SNP allele target is about 52.degree.
C. at 500 nM, assuming a 150 nM target concentration; and the
melting temperature of the OFF probe hybridized to the mismatched C
SNP allele target is about 41.degree. C. at 500 nM, assuming the
same target concentration. The ON probe consisted of a linear probe
labeled at the 5' end with a Quasar 670 fluorophore and at the 3'
end with a BHQ2 quencher. This probe was designed to have a melting
temperature of 62.degree. C. and to hybridize adjacent to the OFF
probe binding site such that upon binding to the PCR product, the
fluorophore moiety of the ON probe resides next the BHQ2 quencher
from the OFF probe. Reaction components and conditions were as
follows:
TABLE-US-00014 Limiting Primer: (SEQ ID No. 72) 5'
GTGAAGGGATTACAAGGCGTGAGGCAC 3', Tm = 71.2 Excess Primer: (SEQ ID
No. 73) 5' GGACTACTTAGCCTCCAATTCAC Tm - 66.2 ON Probe: (SEQ ID No.
74) QUASAR 670--5' CGATATTTATTCCAACATACACCGTG 3' BHQ 2, Tm = 62.5
OFF Probe: (SEQ ID No. 75) 5' CCGATCAAAATTTATATT 3' BHQ 2, Tm =
51.6 (the underlined nucleotide corresponds to the SNP position)
PrimeSafe 060: (SEQ ID No. 76)
5'-DABCYL-CGCGGCGTCAGGCATATAGGATACCGGGAC AGACGCCG CG-DABCYL-3'
PrimeSafe 002: (SEQ ID No. 77)
5'-DABCYL-CGTAATTATAAT-C3spacer-ATTATAATTACG DABCYL-3'
Primesafe (Rice et al. Nat Protoc. 2007; 2(10):2429-38., herein
incorporated by reference in its entirety), is a PCR additive that
maintains the fidelity of amplification over a broad range of
target concentrations by suppressing mis-priming throughout the
reaction. Methods: Genomic DNAs of known genotypes for the
rs3731239 SNP site (C/T alleles) were obtained from the Coriell
Cell Repository (Camden, N.J.; DNA sample NA10860--homozygous TT
alleles; DNA sample NA 10854--heterozygous CT alleles; DNA sample
NA07348 homozygous CC alleles). LATE-PCR amplification, in
triplicate, of the genomic DNA segment containing the above SNP
site from each of the above DNA samples was done in a 25 .mu.l
reaction consisting of 1.times.PCR buffer (Invitrogen, Carlsbad,
Calif.), 3 mM MgCl.sub.2, 250 nM dNTP, 1 .mu.l M excess primer, 50
nM limiting primer, 500 nM OFF probe, 200 nM ON probe, 1.25 units
Platinum Taq DNA polymerase (Invitrogen, Carlsbad, Calif.) and 1000
genomes equivalent of genomic DNA (6 ng). For this experiment the
amplification reactions were optimized by including in the reaction
mixture a combination of reagents intended to avoid mispriming and
to reduce scatter among replicate samples. In this experiment,
reagents for reducing mispriming and improving reproducibility were
included, according to issued U.S. Pat. No. 7,517,977 and
corresponding international patent application WO 2006/044995. The
reaction mixture included 25 nM PrimeSafe 060 and 300 nM PrimeSafe
002. A control reaction consisted of no template controls (NTC
samples).
LATE-PCR amplification was carried out in a Biorad IQ5 Real-Time
PCR Detection System. The amplification conditions were 95.degree.
C. for 3 minutes, then 70 cycles of 95.degree. C. for 10 seconds,
64.degree. C. for 10 seconds, 72.degree. C. for 20 seconds. The
reaction temperature was then brought to 30.degree. C. at a rate of
1.degree. C./min. Fluorescent signals were collected at every
1.degree. C. as each sample was next heated at a rate of 1.degree.
C. per 30 seconds from 30.degree. C. to 80.degree. C.
Raw fluorescent signals collected from each amplification reaction
at every temperature were exported to Microsoft Excel. The
fluorescent signals for any given melting curve were then
normalized as follows: (a) the fluorescent signals at each
temperature were first normalized for background fluorescence by
dividing the fluorescent signal value at each temperature by the
fluorescent signal value at 66.degree. C. (a temperature at which
the ON probe is not bound to its target); (b) the resulting
fluorescent signal value at each temperature was then subtracted
from the average fluorescent signal value from the NTC samples at
that temperature; and (c) the resulting fluorescent signal values
were then normalized to the fluorescent signal value at the
temperature at which the ON probe is bound to the totality of PCR
products (58.degree. C.) and then normalized to the fluorescent
signal value at the temperature at which the OFF probe is bound to
the totality of PCR products and signals from the ON probe are
turned off (38.degree. C.). The latter was accomplished by
sequentially dividing each fluorescent signal value by the
fluorescent signal values at those two temperatures. FIG. 20 shows
the resulting normalized fluorescent pattern (fluorescence versus
temperature) for the three genotypes. Circle 223 identifies the
samples that were homozygous for the matched allele; circle 224
identifies the samples that were heterozygous; and circle 225
identifies the samples that were homozygous for the mismatched
allele. Each genotype generated a characteristic fluorescent
signature. In FIG. 20 the temperature of 49.degree. C. is the
temperature at which the normalized fluorescence signals from the
three genotypes exhibited the greatest difference. Values at that
single temperature were judged to be statistically adequate to
distinguish DNA samples homozygous and heterozygous for the
rs3731239 SNP site.
FIG. 21 presents the first derivative of the fluorescent patterns
of FIG. 20. Circle 226 identifies the samples that were homozygous
for the matched alleles; circle 227 identifies the samples that
were heterozygous, and circle 228 identifies the samples that were
homozygous for the mismatched allele. Positive first derivative
values illustrate the binding of the ON probe, which is the same
for all genotypes. Negative first derivative values illustrate the
allele-specific binding of the OFF probe. Thus, curves 226 for the
homozygous samples containing the matched allele show a single
negative peak of high Tm; curves 228 for the homozygous samples
containing the mismatched alleles show a single negative peak of
lower Tm; and curves 227 for the heterozygous samples containing
both alleles show two negative peaks corresponding to the negative
peaks the matched and mismatched SNP alleles.
Example 10
SPA Typing of MRSA Samples
Recently, DNA sequencing of the polymorphic X, or short sequence
repeat (SSR), region of the protein A gene (spa) has been proposed
for the typing of S. aureus. The polymorphic X region consists of a
variable number of 24-bp repeats and is located immediately
upstream of the region encoding the C-terminal cell wall attachment
sequence. The existence of well-conserved regions flanking the X
region coding sequence in spa allows the use of primers for PCR
amplification and direct sequence typing. This example describes
the use of a LATE-PCR assay using ON/OFF probes to distinguish
strains of S. aureus based on the X repeat region and to create a
signature library where different strains can be identified. The
assay of this example was designed and tested using a panel of
twelve sequenced MRSA samples. The sequence analysis of the samples
is given in Table 6.
TABLE-US-00015 TABLE 6 MRSA Sample Sequences Species type spa
repeat sequence* COL I YHGFMBQBLO (SEQ ID No. 78) N315 II
TJMBMDMGMK (SEQ ID No. 79) 85/2082 III WGKAOMQ (SEQ ID No. 80) CA05
IVA A2AKEMBKB (SEQ ID No. 81) 8/6-3P IVB YHGFMBQBLO (SEQ ID No. 82)
Q2314 IVC TJMBMDMGGMK (SEQ ID No. 83) JCSC4469 IVD TJMBMDMGMK (SEQ
ID N. 84) AR43/3330.1 IVE YMBQBLO (SEQ ID No. 85) HAR22 IVH
TJEJNF2MNF2MOMOKR (SEQ ID No. 86) WIS V A2AKBEKBKB (SEQ ID No. 87)
HDE288 VI TJMBDMGMK (SEQ ID No. 88) BK20781 VIII YHGFC2BQBLO (SEQ
ID No. 89) *To make the notation shorter in the table, letter codes
ending with "1" in the standard nomenclature have been simplified
by omitting this numeral.
The DNA target to be utilized for designing the assay was selected
by examining the sequences of the species in the panel shown in
Table 6 and in NCBI Genbank for a sequence that fits the criteria
described above and whose variable region includes sufficient
differences among the target species. By this method, a 507 base
pair region was selected, namely, nucleotides 262-768, of the spa
gene of S. aureus, MRSA252, NCBI Genbank reference number
NC_002952. The gene sequence for the above region of MRSA252 were
downloaded into a computer program for primer and probe design.
Visual OMP (DNASoftware, Inc., Ann Arbor, Mich., USA) was used for
the assay design software. Using the design software, a primer set
and a set of probes was designed comprising one signaling ("ON")
probe and one quencher ("OFF") probe. Nucleic acid sequences for
the repeat codes were obtained from Shopsin et al., J. Clinical
Microbiology, November 1999, pages 3556-3563. For alignment of
potential probe sequences to various repeat sequences, reverse
complements of repeat sequences obtained from that paper were
used.
To reach a final design of primers and probes, the initial design
was treated as prospective. Several of the sequences were selected
as design sequences, which were run through another alignment to
confirm the appropriate homology with the target sequence.
Using the foregoing method, a pair of primers was designed, which
bracket the target sequence, and a pair of probes. The sequences of
the primers and probes, plus the MRSA 252 target sequence, are
given below. The excess primer is a consensus primer. For this
example two probes were used: an ON probe that has a consensus
sequence that matches the largest number of bases (19 of 24) in the
possible repeat segments shown in Shopsin et al. and an OFF probe
with a consensus sequence that matches the next largest number of
bases (15 of 24) in the possible repeat sequences shown in Shopsin
et al. The probes will compete hybridize adjacently to one another
and result in a signature in an anneal curve done after the
amplification. The anneal signature can then be compared to a
library of signatures and their respective strain of S. aureus.
Each of the signaling, or "ON", probes is a molecular beacon probe
having a stem of two nucleotides, with addition of nucleotides that
are not complementary to the target sequences as needed (such added
nucleotides being bolded in the sequences for identification). The
signaling probe has a Quasar 670 fluorophore (Biosearch
Technologies, Novato, Calif., USA) on one end and a BHQ2 quencher
on the other end. The one quencher probe has a BHQ2 quencher but no
fluorophore. The stated primer and probe Tm's are the calculated
concentration-adjusted melting temperatures used for LATE-PCR. The
probe Tm's are Tm's against perfectly complementary sequences.
TABLE-US-00016 Limiting Primer: (SEQ ID No. 90)
5'-CTGTATCACCAGGTTTAACGACATGTACTCCGT, Tm = 71.0 Excess Primer: (SEQ
ID No. 91) 5'-GCTAAATGATGCTCAAGCACCAA, Tm = 67.2 Target 507,
262-768 (MRSA 252): (SEQ ID No. 92) 5'-
CTGTATCACCAGGTTTAACGACATGTACTCCGTTGCCGTCTTCTTTACC
AGGCTTGTTGCCATCTTCTTTACCAGGCTTGTTGCCATCTTCTTTACCAG GCTTGTT
GCCATCTTCTTTACCAGGCTTGTTGCCGTCTTCTTTACCAGGTTTGTTG CCATC
TTCTTTGCCAGGTTTTTTGTTGTCTTCTTTACCAGGTTTGTTGCCGTCTT
CTTTGCCAGGTTTTTTGTTGTCTTCTTTACCAGGTTTGTTGCCGTCTTCT
TTACCAGGCTTGTTGTTGTCTTCTTTGCCAGGCTTGTTGTTGTCTTCCTC TTTTGGTGCTTGA
GCATCGTTTAGCTTTTTAGCTTCTGCTAAAATTTCTTTGCTCACTGAAG
GATCGTCTTTAAGGCTTTGGATGAAGCCGTTACGTTGTTCTTCAGTTAA
GTTAGGTAAATGTAAAATTTCATAGAAAGCATTTTGTTGTTCTTTGTTGA
ATTTGTTGTCAGCTTTTGGTGCTTGTGCATCATTTAGC spa ON Probe: (SEQ ID No.
93) 5'-Quasar 670-AACCAGGCTTGTTGTTGTCTTCTTT-BHQ2, Tm = 66.6 spa Off
Probe: (SEQ ID No. 94) 5'-AAGCCAGGTTTTTTGCCATCTTCTTT-BHQ2, Tm =
59.8
Twenty-five .mu.L LATE-PCR reaction mixtures including a single
bacterial genomic DNA target contained 10.times.PCR Buffer 1.times.
(final concentration), 10 mM dNTPs 250 .mu.M (final concentration),
50 mM Mg.sup.++3 mM (final concentration), 10 .mu.M Limiting Primer
50 nM (final concentration), 100 .mu.M Excess Primer 1000 nM (final
concentration), 10 .mu.M signaling probe 100 nM (final
concentration), 10 .mu.M of quencher probe 100 nM (final
concentration), 1.25 Units Platinum Taq DNA polymerase, and
10.sup.6 bacterial genomic DNA starting copies. A Taq
polymerase-only control (NTC) containing Taq polymerase and all of
the above reagents but no genomic DNA was also amplified.
Amplification and detection of three replicate samples of each
target and each control were performed with a Bio Rad (Hercules,
Calif., USA) IQ5 real-time thermocycler using the following
protocol: denaturation at 95.degree. C. for three minutes, followed
by 50 cycles of 95.degree. C. for 10 seconds, 65.degree. C. for 15
seconds and 72.degree. C. for 45 seconds. Following amplification a
melt curve was generated starting at 25.degree. C. and progressing
to 80.degree. C. in one-degree steps of thirty seconds each and
then an anneal curve was generated starting at 80.degree. C. and
progressing to 25.degree. C. in one degree steps of thirty seconds
each, with data acquisition of Quasar 670 fluorescence at each
step. The various first derivative anneal curves are shown in FIG.
22. Fluorescence intensities were normalized to 75.degree. C. and
with background fluorescence of the NTC control at 75.degree. C.
subtracted, and then the highest fluorescent value normalized to
1.0. FIG. 22 presents the first-derivative annealing curves for the
samples tested. In FIG. 22, circle 229 is the NTC, circle 230 is
HAR22IVH, circle 231 is AR43/3330.1IVE, circle 232 is CA05IVA,
circle 233 is WISV, circle 234 is Q2314IVC, circle 235 is HDE288VI,
circle 236 are N315II and JCSC4469IVD, circle 237 is 85/2082111,
circle 238 are COLI and 8/6-3PIVB, and circle 239 is
BK20781VIII.
Table 6 above lists the spa types of the twelve MRSA samples that
were tested. Some of the samples have the same spa types, others
have similar spa types, and still others have very different spa
types. Results in FIG. 22 showed the expected differentiation and
definition of each spa type. When spa types were expected to be the
same, the same signature appeared, see COL1 and 8/6-3P IVB; and
N315 and JCSC4469 IVD.
The scope is not to be limited by the specific features and
embodiments described above. Various modifications of these
embodiments can be made without departing from the the inventive
concepts described herein. Such modifications are intended to fall
within the scope of the appended claims.
SEQUENCE LISTINGS
1
90131DNAArtificial SequenceSynthetic 1ctccagccag gcacgctcac
gtgacagacc g 31224DNAArtificial SequenceSynthetic 2ccggtggtcg
ccgcgatcaa ggag 243150DNAMycobacterium tuberculosis 3ccggtggtcg
ccgcgatcaa ggagttcttc ggcaccagcc agctgagcca attcatggac 60cagaacaacc
cgctgtcggg gttgacccac aagcgccgac tgtcggcgct ggggcccggc
120ggtctgtcac gtgagcgtgc cgggctggag 1504150DNAMycobacterium
tuberculosis 4ccggtggtcg ccgcgatcaa ggagttcttc ggcaccagcc
agctgagcca attcatggtc 60cagaacaacc cgctgtcggg gttgacccac aagcgccgac
tgtcggcgct ggggcccggc 120ggtctgtcac gtgagcgtgc cgggctggag
1505150DNAMycobacterium tuberculosis 5ccggtggtcg ccgcgatcaa
ggagttcttc ggcaccagcc agctgagcca attcatggac 60cagaacaacc cgctgtcggg
gttgacccac aagcgccgac tgttggcgct ggggcccggc 120ggtctgtcac
gtgagcgtgc cgggctggag 150616DNAArtificial SequenceSynthetic
6ctggttggtg cagaag 16723DNAArtificial SequenceSynthetic 7tcaggtccat
gaattggctc aga 23812DNAArtificial SequenceSynthetic 8cagcgggttg tt
12924DNAArtificial SequenceSynthetic 9atgcgcttgt ggatcaaccc cgat
241023DNAArtificial SequenceSynthetic 10aagccccagc gccgacagtc gtt
231112DNAArtificial SequenceSynthetic 11acagaccgcc gg
121224DNAArtificial SequenceSynthetic 12accagggctg ggccatgcgc acca
241321DNAArtificial SequenceSynthetic 13ggaccgcagc cacgccaagt c
2114113DNAMycobacterium tuberculosis 14ggaccgcagc cacgccaagt
cggcccggtc ggttgccgag accatgggca actaccaccc 60gcacggcgac gcgtcgatct
acgacagcct ggtgcgcatg gcccagccct ggt 11315113DNAMycobacterium
tuberculosis 15ggaccgcagc cacgccaagt cggcccggtc ggttgccgag
accatgggca actaccaccc 60gcacggcgac gtgtcgatct acgacagcct ggtgcgcatg
gcccagccct ggt 1131610DNAArtificial SequenceSynthetic 16cgaccgggcc
101721DNAArtificial SequenceSynthetic 17aacccatggt ctcggcaact t
211822DNAArtificial SequenceSynthetic 18aatcgccgtg cgggtggtag tt
221919DNAArtificial SequenceSynthetic 19gctgtcgtag atcgacgcg
192037DNAArtificial SequenceSynthetic 20agcgcccact cgtagccgta
caggatctcg aggaaac 372128DNAArtificial SequenceSynthetic
21tcttgggctg gaagagctcg tatggcac 2822139DNAMycobacterium
tuberculosis 22gcttgggctg gaagagctcg tatggcaccg gaaccggtaa
ggacgcgatc accagcggca 60tcgaggtcgt atggacgaac accccgacga aatgggacaa
cagtttcctc gagatcctgt 120acggctacga gtgggagct
13923139DNAMycobacterium tuberculosis 23gcttgggctg gaagagctcg
tatggcaccg gaaccggtaa ggacgcgatc accaccggca 60tcgaggtcgt atggacgaac
accccgacga aatgggacaa cagtttcctc gagatcctgt 120acggctacga gtgggagct
13924139DNAMycobacterium tuberculosis 24gcttgggctg gaagagctcg
tatggcaccg gaaccggtaa ggacgcgatc accaacggca 60tcgaggtcgt atggacgaac
accccgacga aatgggacaa cagtttcctc gagatcctgt 120acggctacga gtgggagct
1392521DNAArtificial SequenceSynthetic 25aagtgatcgc gtccttacct t
212616DNAArtificial SequenceSynthetic 26gacctcgatg cagctg
1627150DNAMycobacterium tuberculosis 27ccggtggtcg ccgcgatcaa
ggagttcttc ggcaccagcc agctgagcca attcatggac 60cagaacaacc cgctgtcggg
gttgaccgac aagcgccgac tgtcggcgct ggggcccggc 120ggtctgtcac
gtgagcgtgc cgggctggag 1502836DNAArtificial SequenceSynthetic
28ggttatacct agtataattg gtggttttgg taattg 362936DNAArtificial
SequenceSynthetic 29ggttatacct agtataattg gtggttttgg taactg
363036DNAArtificial SequenceSynthetic 30ggttatacct agtataattg
gtggttttgg caattg 363136DNAArtificial SequenceSynthetic
31actaggatca aaaaaagaag tattaaaatt acgatc 3632460DNACaenorhabditis
elegans 32tcttggatca aaaaatgaag tatttaaatt acgatcagtt aacaacatag
taatagcccc 60tgctaaaacc ggtagagata aaaccagtaa aaacactgtt acaaatacag
ttcaaacaaa 120taaagttata tgttctaatg aaatagaact tctacgtaaa
tttttagtag tacacataaa 180attaatacca cctaagatag atcttaaccc
tgctgcatgt aaactaaaaa tagctaaatc 240tactctactt ccagggtgcc
ccattgttct taaaggtggg tagactgttc acctagtccc 300acaacctata
tctacaaaac aagcatctaa aattaataat atagatgtag gtaataacca
360aaatcttaaa ttatttaaac gtggaaatct tatatcaggt gctcctaaca
taagtggtaa 420taatcagtta ccaaaaccac cgattatagt aggtattacc
46033450DNASteinernema feltiaemisc_feature(437)..(450)n is a, c, g,
or t 33tctaggatca aaaaaagaag tatttaaatt acggtctgta agaagtatag
taattgcccc 60agctaaaacc ggtaaagaaa gaacaagaag gaaaactgta acaaaaacag
ttcaaacaaa 120aagactcata tgctctaaag aaatagagct tctacgaaga
ttcttagtag tacatataaa 180attaatagcc cccaaaatag agcttacacc
agcacaatga agactaaaaa tagctaaatc 240aaccctgttt ccaggatggc
ctaaagtact taaaggagga taaacagttc aactagtacc 300acaccctgta
tctacaaaac aagcatctaa aattaataat atagcagtgg gtaataacca
360aaaacttaaa ttatttaaac gaggaaatct tatatccgga gcaccaagaa
ggaactaatc 420aatttccaaa tcctccnnnn nnnnnnnnnn 4503440DNAArtificial
SequenceSynthetic 34aatattacct ttgatgttag gggctcctga tataagtttt
403545DNAArtificial SequenceSynthetic 35atcctcgttt aaataattta
agtttttgat tattacctac ttcat 453647DNAArtificial SequenceSynthetic
36tttgttttgt tgttgggatt cttgttttgt tgatataggt ggtggaa
473735DNAArtificial SequenceSynthetic 37aactggttga actgtttacc
ctcctttaag aactt 353845DNAArtificial SequenceSynthetic 38aagtaggtca
tcctggtagt actgtagatt ttgttatttt tactt 453938DNAArtificial
SequenceSynthetic 39atgcatggtg ctggttttag ttctattttg ggtgctat
384042DNAArtificial SequenceSynthetic 40attaatttta tgggtactac
tgttaagaat ctgcgtagtt at 424143DNAArtificial SequenceSynthetic
41ttctatttct ttggaacata tgagtctgtt tgtttggact gaa
434236DNAArtificial SequenceSynthetic 42tttttgtgac tgtttttttg
ttggttctgt ctctaa 364340DNAArtificial SequenceSynthetic
43ttcctgtttt aggtggggct attactatat tgttaactaa 404425DNAArtificial
SequenceSynthetic 44ccagactcct acgggaggca gcagt 254521DNAArtificial
SequenceSynthetic 45gtattaccgc ggctgctggc a 214623DNAArtificial
SequenceSynthetic 46aaggggaata ttgcacaatg gtt 234724DNAArtificial
SequenceSynthetic 47aagcgaaagc ctgatgcagc catt 244821DNAArtificial
SequenceSynthetic 48tagccgcgtg tgtgaagaat a 214927DNAArtificial
SequenceSynthetic 49ttggccttcg gattgtaaag cacttaa
275019DNAArtificial SequenceSynthetic 50tattagtagg gaggaagta
195129DNAArtificial SequenceSynthetic 51ttatatgtgt aagtaactgt
gcacatcaa 295216DNAArtificial SequenceSynthetic 52ttgacgttac ccgcaa
165326DNAArtificial SequenceSynthetic 53ttgaagaagc accggctaac
tccgaa 265425DNAArtificial SequenceSynthetic 54ccagactcct
acgggaggca gcagt 255529DNAArtificial SequenceSynthetic 55tggactacca
gggtatctaa tcctgtttg 295621DNAArtificial SequenceSynthetic
56atagggtgcg agcgttaatc t 215725DNAArtificial SequenceSynthetic
57aaggatttac tgggcgtaaa gcgtt 255824DNAArtificial SequenceSynthetic
58ttgcgtaggc ggcttattaa gtaa 245924DNAArtificial SequenceSynthetic
59aacggatgtg aaatccccga gctt 246022DNAArtificial SequenceSynthetic
60taacttggga attgcattcg ta 226123DNAArtificial SequenceSynthetic
61atactggtga gctagagtat gat 236220DNAArtificial SequenceSynthetic
62gaagaggatg gtagaattcc 206323DNAArtificial SequenceSynthetic
63taggtgtagc ggtgaaatgc gta 236423DNAArtificial SequenceSynthetic
64aaggggaata ttgcacaatg gtt 236524DNAArtificial SequenceSynthetic
65aagcgaaagc ctgatgcagc catt 246621DNAArtificial SequenceSynthetic
66tagccgcgtg tgtgaagaat a 216727DNAArtificial SequenceSynthetic
67ttggccttcg gattgtaaag cacttaa 276819DNAArtificial
SequenceSynthetic 68tattagtagg gaggaagta 196929DNAArtificial
SequenceSynthetic 69ttatatgtgt aagtaactgt gcacatcaa
297016DNAArtificial SequenceSynthetic 70ttgacgttac ccgcaa
167126DNAArtificial SequenceSynthetic 71ttgaagaagc accggctaac
tccgaa 267227DNAArtificial SequenceSynthetic 72gtgaagggat
tacaaggcgt gaggcac 277323DNAArtificial SequenceSynthetic
73ggactactta gcctccaatt cac 237426DNAArtificial SequenceSynthetic
74cgatatttat tccaacatac accgtg 267518DNAArtificial
SequenceSynthetic 75ccgatcaaaa tttatatt 187640DNAArtificial
SequenceSynthetic 76cgcggcgtca ggcatatagg ataccgggac agacgccgcg
407724DNAArtificial SequenceSyntheticmisc_feature(12)..(13)The
residues at these positions are linked to a C3 spacer. 77cgtaattata
atattataat tacg 247833DNAArtificial SequenceSynthetic 78ctgtatcacc
aggtttaacg acatgtactc cgt 337923DNAArtificial SequenceSynthetic
79gctaaatgat gctcaagcac caa 2380509DNAStaphylococcus aureus
80ctgtatcacc aggtttaacg acatgtactc cgttgccgtc ttctttacca ggcttgttgc
60catcttcttt accaggcttg ttgccatctt ctttaccagg cttgttgcca tcttctttac
120caggcttgtt gccgtcttct ttaccaggtt tgttgccatc ttctttgcca
ggttttttgt 180tgtcttcttt accaggtttg ttgccgtctt ctttgccagg
ttttttgttg tcttctttac 240caggtttgtt gccgtcttct ttaccaggct
tgttgttgtc ttctttgcca ggcttgttgt 300tgtcttcctc ttttggtgct
tgagcatcgt ttagcttttt agcttctgct aaaatttctt 360tgctcactga
aggatcgtct ttaaggcttt ggatgaagcc gttacgttgt tcttcagtta
420agttaggtaa atgtaaaatt tcatagaaag cattttgttg ttctttgttg
aatttgttgt 480cagcttttgg tgcttgtgca tcatttagc 5098125DNAArtificial
SequenceSynthetic 81aaccaggctt gttgttgtct tcttt 258226DNAArtificial
SequenceSynthetic 82aagccaggtt ttttgccatc ttcttt
2683232DNAKlebsiella pneumonia 83gtccagactc ctacgggagg cagcagtggg
gaatattgca caatgggcgc aagcctgatg 60cagccatgcc gcgtgtgtga agaaggcctt
cgggttgtaa agcactttca gcggggagga 120aggcggtgag gttaataacc
tcatcgattg acgttacccg cagaagaagc accggctaac 180tccgtgccag
cagccgcggt aatacggagg gtgcaagcgt taatcggaat ta
23284232DNAEnterobacter aerogenes 84gtccagactc ctacgggagg
cagcagtggg gaatattgca caatgggcgc aagcctgatg 60cagccatgcc gcgtgtatga
agaaggcctt cgggttgtaa agtactttca gcgaggagga 120aggcattgtg
gttaataacc gcagtgattg acgttactcg cagaagaagc accggctaac
180tccgtgccag cagccgcggt aatacggagg gtgcaagcgt taatcggaat ta
23285233DNAAcinetobacter baumannii 85gcccagactc ctacgggagg
cagcagtggg gaatattgga caatgggggg aaccctgatc 60cagccatgcc gcgtgtgtga
agaaggcctt atggttgtaa agcactttaa gcgaggagga 120ggctacttta
gttaatacct agagatagtg gacgttactc gcagaataag caccggctaa
180ctctgtgcca gcagccgcgg taatacagag ggtgcgagcg ttaatcggat tta
23386232DNAPseudomonas aeruginosa 86gtccagactc ctacgggagg
cagcagtggg gaatattgga caatgggcga aagcctgatc 60cagccatgcc gcgtgtgtga
agaaggtctt cggattgtaa agcactttaa gttgggagga 120agggcagtaa
gttaatacct tgctgttttg acgttaccaa cagaataagc accggctaac
180ttcgtgccag cagccgcggt aatacgaagg gtgcaagcgt taatcggaat ta
23287232DNAStaphylococcus aureus 87gtccagactc ctacgggagg cagcagtagg
gaatcttccg caatgggcga aagcctgacg 60gagcaacgcc gcgtgagtga tgaaggtctt
cggatcgtaa aactctgtta ttagggaaga 120acatatgtgt aagtaactgt
gcacatcttg acggtaccta atcagaaagc cacggctaac 180tacgtgccag
cagccgcggt aatacgtagg tggcaagcgt tatccggaat ta
23288232DNAStaphylococcus epidermidis 88gtccagactc ctacgggagg
cagcagtagg gaatcttccg caatgggcga aagcctgacg 60gagcaacgcc gcgtgagtga
tgaaggtctt cggatcgtaa aactctgtta ttagggaaga 120acaaatgtgt
aagtaactat gcacgtcttg acggtaccta atcagaaagc cacggctaac
180tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggaat ta
23289232DNAEnterococcus faecalis 89gcccagactc ctacgggagg cagcagtagg
gaatcttcgg caatggacga aagtctgacc 60gagcaacgcc gcgtgagtga agaaggtttt
cggatcgtaa aactctgttg ttagagaaga 120acaaggacgt tagtaactga
acgtcccctg acggtatcta accagaaagc cacggctaac 180tacgtgccag
cagccgcggt aatacgtagg tggcaagcgt tgtccggatt ta
23290540DNAAcinetobacter baumannii 90cactgggact gagacacggc
ccagactcct acgggaggca gcagtgggga atattggaca 60atggggggaa ccctgatcca
gccataccgc gtgtgtgaag aaggccttat ggttgtaaag 120cactttaagc
gaggaggagg ctactttagt taatacctag ggatagtgga cgttactcgc
180agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg
tgcgagcgtt 240aatcggattt actgggcgta aagcgtgcgt aggcggctta
ttaagtcgga tgtgaaatcc 300ccgagcttaa cttgggaatt gcattcgata
ctggtgagct agagtatggg agaggatggt 360agaattccag gtgtagcggt
gaaatgcgta gagatctgga ggaataccga tggcgaaggc 420agccatctgg
cctaatactg acgctgaggt acgaaagcat ggggagcaaa caggattaga
480taccctggta gtccatgccg taaacgatgt ctactagccg ttggggcctt
tgaggcttta 540
* * * * *
References